TW201836623A - Treatment of retinal degeneration using progenitor cells - Google Patents
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本申請案係2015年12月4日提出申請之美國專利申請案第14/960,006號之部分延續申請案,其主張2014年12月16日提出申請之美國臨時申請案第62/092,658號及2015年10月2日提出申請之美國臨時申請案第62/236,732號之權益,上述各案之全部內容以引用方式併入本文中。 This application is a partial continuation of US Patent Application No. 14 / 960,006 filed on December 4, 2015, and it claims US Provisional Application No. 62 / 092,658 filed on December 16, 2014 and 2015 The rights and interests of US Provisional Application No. 62 / 236,732 filed on October 2, 2003, the entire contents of the above cases are incorporated by reference.
本發明係關於用於眼科疾病及病症的基於細胞或再生性療法的領域。詳言之,本發明提供使用前驅細胞(諸如臍帶組織衍生細胞及胎盤組織衍生細胞)及由該些細胞製備之條件培養基來再生或修復眼細胞及組織之方法及組成物。 The present invention relates to the field of cell-based or regenerative therapies for ophthalmic diseases and disorders. In detail, the present invention provides methods and compositions for regenerating or repairing eye cells and tissues using precursor cells (such as umbilical cord tissue-derived cells and placental tissue-derived cells) and conditioned medium prepared from these cells.
眼睛是一個複雜且敏感的身體器官,會罹患許多疾病及其他影響其正常發揮功能之能力的有害病狀。許多該些病狀與特定眼細胞及由該些細胞組成之組織的損傷或變性相關聯。舉例而言,視神經及視網膜之疾病及變性病狀為全世界失明之首要原因。 角膜、水晶體及相關眼組織之損傷或變性為全世界視力喪失之另一顯著原因。 The eye is a complex and sensitive body organ that can suffer from many diseases and other harmful conditions that affect its ability to function normally. Many of these pathologies are associated with damage or degeneration of specific eye cells and tissues composed of these cells. For example, diseases and degenerative conditions of the optic nerve and retina are the leading causes of blindness worldwide. Damage or degeneration of the cornea, crystals and related eye tissues is another significant cause of vision loss worldwide.
視網膜含有七層交替細胞及將光信號轉化成神經信號之過程。視網膜光受體與鄰近視網膜色素上皮(RPE)形成功能單元,其在許多病症中由於遺傳突變或環境條件(包括年齡)而變得不平衡。此導致光受體經由細胞凋亡或繼發性變性而損失,從而導致視力之進行性劣化並在一些情況下導致失明(有關綜述參見例如,Lund,R.D.等人,Progress in Retinal and Eye Research,2001;20:415-449)。具有此模式之兩類眼病症為年齡相關性黃斑變性(AMD)及色素性視網膜炎(RP)。 The retina contains seven layers of alternating cells and the process of converting light signals into neural signals. The retinal photoreceptor forms a functional unit with the adjacent retinal pigment epithelium (RPE), which in many disorders becomes unbalanced due to genetic mutations or environmental conditions (including age). This leads to the loss of photoreceptors through apoptosis or secondary degeneration, leading to progressive deterioration of vision and in some cases blindness (for a review, see, for example, Lund, RD et al., Progress in Retinal and Eye Research , 2001; 20: 415-449). Two types of eye disorders with this pattern are age-related macular degeneration (AMD) and retinitis pigmentosa (RP).
AMD在美國是50歲或年齡更大之人口中視力喪失之最普遍原因且其盛行率隨年齡升高。AMD之原發性病症似乎係因RPE功能異常及在布魯赫氏(Bruch's)膜中之變化所致,其特徵在於特別是脂質沉積、蛋白質交聯及營養素滲透性降低(參見Lund等人,2001見前文)。多種要素可導致黃斑變性,包括遺傳構成、年齡、營養、吸煙、及暴露於陽光或其他氧化應力。非滲出性、或「乾性(dry)」形式之AMD佔AMD病例之90%;其他10%為滲出性、新生血管形式(「濕性(wet)」AMD)。在乾性AMD患者中,視網膜色素上皮(RPE)之逐漸消失導致環繞狀之萎縮區域。因為光受體損失跟隨在RPE消失之後,所以受影響之視網膜區域僅剩極少或完全無視覺功能。 AMD is the most common cause of vision loss in the 50-year-old or older population in the United States and its prevalence increases with age. The primary disease of AMD appears to be due to abnormal RPE function and changes in Bruch's membranes, which is characterized by, inter alia, lipid deposition, protein cross-linking, and reduced nutrient permeability (see Lund et al., See above in 2001). Various factors can cause macular degeneration, including genetic makeup, age, nutrition, smoking, and exposure to sunlight or other oxidative stress. Non-exudative, or "dry" forms of AMD account for 90% of AMD cases; the other 10% are exudative, neovascularized forms ("wet" AMD). In patients with dry AMD, the gradual disappearance of retinal pigment epithelium (RPE) results in surrounding atrophic areas. Because the loss of photoreceptors follows the disappearance of RPE, there is little or no visual function left in the affected retinal area.
AMD之當前療法涉及諸如例如雷射療法及醫藥介入之程序。雷射波束藉由轉移熱能來破壞黃斑下之滲漏血管,以延緩視力喪失之速率。雷射療法之缺點在於,由波束遞送之高熱能亦破壞附近的健康組織。Neuroscience第4版(Purves,D,等人2008)指出「目前沒有針對乾性AMD之治療」。 AMD's current therapies involve procedures such as, for example, laser therapy and medical intervention. The laser beam transfers heat energy to destroy the leaking blood vessels under the macula to delay the rate of vision loss. The disadvantage of laser therapy is that the high thermal energy delivered by the beam also destroys nearby healthy tissue. The fourth edition of Neuroscience (Purves, D, et al. 2008) states that "there is currently no treatment for dry AMD."
RPE移植在人類中尚未成功。舉例而言,Zarbin,M,2003指出「在正常老化情況下,人類布魯赫氏膜(尤其在黃斑下區域中)經歷許多改變(例如,厚度增大、ECM及脂質之沉積、蛋白質之交聯、高級糖化終產物之非酶形成)。此等改變及由於AMD之額外改變可能降低ECM配體(例如,層黏蛋白、纖連蛋白素、及膠原蛋白IV)之生物可利用性,且造成AMD眼睛中極為不良的RPE細胞存活。因此,儘管人類RPE細胞表現附著於此等ECM分子所需之整合素,但是老化的黃斑下人類布魯赫氏膜上之RPE細胞存活仍然受損。」(Zarbin,MA,Trans Am Ophthalmol Soc,2003;101:493-514)。 RPE transplantation has not been successful in humans. For example, Zarbin, M, 2003 pointed out that "under normal aging, human Bruch's membrane (especially in the submacular region) undergoes many changes (eg, increased thickness, deposition of ECM and lipids, protein exchange Non-enzymatic formation of the final product of oligomerization and advanced glycation. These changes and additional changes due to AMD may reduce the bioavailability of ECM ligands (eg, laminin, fibronectin, and collagen IV), and Causes extremely poor RPE cell survival in AMD eyes. Therefore, although human RPE cells exhibit the integrin required to attach to these ECM molecules, the survival of RPE cells on human Bruch's membrane under the aging macula is still impaired. "(Zarbin, MA, Trans Am Ophthalmol Soc, 2003; 101: 493-514).
色素性視網膜炎主要被認為是一種遺傳性疾病,其中超過100種突變與光受體損失相關聯(參見Lund等人,2001,見前文)。儘管大部分突變以光受體為目標,但有些突變直接影響RPE細胞。綜上,此等突變影響諸如光受體與RPE細胞之間的分子運輸及光傳導之過程。 Retinitis pigmentosa is primarily considered to be an inherited disease in which more than 100 mutations are associated with loss of photoreceptors (see Lund et al., 2001, see above). Although most mutations target photoreceptors, some mutations directly affect RPE cells. In summary, these mutations affect processes such as molecular transport and light transmission between photoreceptors and RPE cells.
其他較不常見但亦有損健康的視網膜病變也可涉及導致視力喪失及失明之進行性細胞變性。此等視網膜病變包括例如糖尿病視網膜病變及脈絡膜新生血管膜(CNVM)。 Other less common but also detrimental health retinopathy can also involve progressive cell degeneration that leads to vision loss and blindness. Such retinopathy includes, for example, diabetic retinopathy and choroidal neovascular membrane (CNVM).
用於組織修復及再生之基於幹細胞的療法的出現,對許多前述細胞變性病變及其他眼病症提供具潛力之治療。幹細胞能夠自我更新及分化以產生多種成熟細胞譜系。此類細胞之移植可用來作為重構靶標組織之臨床工具,從而恢復生理及結構功能。幹細胞技術之應用範圍廣泛,包括組織工程化、基因療法遞送、及細胞治療,亦即,經由外源性供應可生產或含有生物治療劑之活細胞或細胞組分來遞送該些劑至靶標位置。(有關綜述,參見例如,Tresco,P.A.等人,Advanced Drug Delivery Reviews,2000,42:2-37)。 The emergence of stem cell-based therapies for tissue repair and regeneration provides potential treatments for many of the aforementioned cell degeneration and other eye disorders. Stem cells are capable of self-renewal and differentiation to produce a variety of mature cell lineages. The transplantation of such cells can be used as a clinical tool to reconstruct target tissues, thereby restoring physiological and structural functions. Stem cell technology has a wide range of applications, including tissue engineering, gene therapy delivery, and cell therapy, that is, the delivery of these agents to target locations through the exogenous supply of live cells or cell components that can produce or contain biotherapeutics . (For a review, see, for example, Tresco, PA et al., Advanced Drug Delivery Reviews , 2000, 42: 2-37).
最近,已顯示產後衍生細胞改善視網膜變性(US 2010/0272803)。皇家外科學院(Royal College of Surgeons,RCS)大鼠具有影響外節吞噬之酪胺酸受體激酶(Mertk)缺陷,從而引起光受體細胞死亡。(Feng W.等人,J Biol Chem.,2002,10:277(19):17016-17022)。發現將視網膜色素上皮(RPE)細胞移植至RCS大鼠之視網膜下空間中會限制光受體損失之進展且保留視覺功能。(US 2010/0272803)。亦已證明,產後衍生細胞可在RCS模型中用於促進光受體救援且因此保留光受體。(US 2010/0272803)。將人類臍帶組織衍生細胞(hUTC)經視網膜下注射至RCS大鼠眼睛中改良視覺銳度且改善視網膜變性(US 2010/0272803;Lund RD等人,Stem Cells.2007;25(3):602-611)。此外,以衍生自hUTC之條件培養基(CM) 進行治療,可使體外失養性RPE細胞恢復對ROS之吞噬。(US 2010/0272803)。 Recently, postpartum-derived cells have been shown to improve retinal degeneration (US 2010/0272803). Royal College of Surgeons (RCS) rats have defects in the tyrosine receptor kinase (Mertk) that affects phagocytosis of the outer segment, causing photoreceptor cell death. (Feng W. et al., J Biol Chem., 2002, 10: 277 (19): 17016-17022). It was found that transplantation of retinal pigment epithelium (RPE) cells into the subretinal space of RCS rats limits the progress of photoreceptor loss and preserves visual function. (US 2010/0272803). It has also been shown that postpartum-derived cells can be used in the RCS model to promote photoreceptor rescue and thus retain photoreceptors. (US 2010/0272803). Human umbilical cord tissue-derived cells (hUTC) were injected subretinally into the eyes of RCS rats to improve visual acuity and improve retinal degeneration (US 2010/0272803; Lund RD et al., Stem Cells. 2007; 25 (3): 602- 611). In addition, treatment with conditioned medium (CM) derived from hUTC can restore the phagocytosis of ROS by in vitro dystrophic RPE cells. (US 2010/0272803).
由吞噬細胞清除細胞凋亡細胞為正常生命不可或缺的一部分,在此過程中之缺陷會對自身耐受性及自體免疫性產生顯著影響(Ravichandran等人,Cold Spring Harb Perspect Biol.,2013,5(1):a008748.doi:10.1101/cshperspect.a008748.Review)。細胞凋亡細胞之辨識及移除主要由職業性吞噬細胞(受體結合病原體以進行吞噬)諸如巨噬細胞、單核細胞、及其他白血球,及非職業性吞噬細胞(吞噬不是主要功能)諸如上皮細胞、RPE細胞、內皮細胞所媒介。迄今為止已識別出許多「吃我(eat me)」信號,包括表面蛋白質之醣基化改變或表面電荷之改變(Ravichandran等人,Cold Spring Harb Perspect Biol.,2013)。磷脂醯絲胺酸(PS)之外化為細胞凋亡之標誌,且為研究最多的「吃我(eat me)」信號(Wu等人,Trends.Cell Biol.,2006,16(4):189-197)。「吃我」信號由吞噬細胞吞沒(engulfment)受體直接辨識(如由PS受體辨識),或間接經由橋分子及輔助受體來辨識(Erwig等人,Cell Death.Differ.,2008;15:243-250)。橋分子乳脂小球EGF因子8(MFG-E8)、生長停滯特異性6(growth arrest-specific 6,Gas6)、蛋白質S、血小板反應蛋白(thrombospondin;TSP)、載脂蛋白H(先前稱為β2-醣蛋白,β2-GPI)全部結合至細胞凋亡細胞表面上之PS。接著,MFG-E8可藉由αvβ3及αvβ5整合素透過其RGD模體辨識(Hanayama等人,Science,2004,304:1147-1150;Borisenko等人,Cell Death Differ.,2004: 11:943-945),Gas6可藉由Axl、Tyro3及Mer家族之受體酪胺酸激酶辨識(Scott等人,Nature,2001;411:207-211),且載脂蛋白H可至β2-GPI受體辨識(Balasubramanian等人,J Bio Chem,1997;272:31113-31117)。其他橋分子與細胞凋亡細胞表面上改變的糖及/或脂質之辨識有關,諸如膠凝素(collectin)家族之成員界面活性蛋白質A及D(Vandivier等人,J Immunol,2002;169:3978-398)。 The removal of apoptotic cells by phagocytes is an indispensable part of normal life. Defects in this process will have a significant impact on self-tolerance and autoimmunity (Ravichandran et al. Cold Spring Harb Perspect Biol. , 2013 , 5 (1): a008748.doi: 10.1101 / cshperspect.a008748.Review). Identification and removal of apoptotic cells are mainly caused by occupational phagocytes (receptors bind pathogens for phagocytosis) such as macrophages, monocytes, and other white blood cells, and non-professional phagocytic cells (phagocytosis is not the main function) such as Mediated by epithelial cells, RPE cells, and endothelial cells. Many "eat me" signals have been identified so far, including changes in glycosylation of surface proteins or changes in surface charge (Ravichandran et al., Cold Spring Harb Perspect Biol., 2013). Phosphatidylserine (PS) is externalized as a sign of apoptosis and is the most studied "eat me" signal (Wu et al., Trends. Cell Biol., 2006 , 16 (4): 189-197). The "eat me" signal is directly recognized by the engulfment receptors of phagocytes (such as by PS receptors), or indirectly via bridge molecules and co-receptors (Erwig et al., Cell Death.Differ. , 2008; 15; : 243-250). Bridge molecule milk fat globule EGF factor 8 (MFG-E8), growth arrest specific 6 (growth arrest-specific 6, Gas6), protein S, thrombospondin (thrombospondin; TSP), apolipoprotein H (formerly known as β2 -Glycoprotein, β2-GPI) all bind to PS on the surface of apoptotic cells. Next, MFG-E8 can be identified through its RGD motif by αvβ3 and αvβ5 integrins (Hanayama et al., Science , 2004, 304: 1147-1150; Borisenko et al., Cell Death Differ. , 2004: 11: 943-945 ), Gas6 can be identified by the receptor tyrosine kinases of the Axl, Tyro3 and Mer families (Scott et al., Nature , 2001; 411: 207-211), and apolipoprotein H can be identified by the β2-GPI receptor ( Balasubramanian et al., J Bio Chem , 1997; 272: 31113-31117). Other bridge molecules are involved in the identification of altered sugars and / or lipids on the surface of apoptotic cells, such as the interface-active proteins A and D of members of the collectin family (Vandivier et al., J Immunol, 2002; 169: 3978 -398).
分子之膠凝素家族隨後係經由其膠原尾部與鈣網伴護蛋白(calreticulin,CRT)之交互作用而經辨識,此繼而藉由吞噬細胞經由低密度脂蛋白(LDL)受體相關蛋白質(LRP-1/CD91)傳導攝取信號(Gardai等人,Cell,2003;115:13-23)。作為另一實例,所識別之第一橋分子為血小板反應蛋白(TSP)-1(Savill等人,J Clin Invest,1992;90:1513-1522),其係作為細胞外基質醣蛋白,且認為其結合至細胞凋亡細胞上之TSP-1結合位點且隨後結合至吞噬細胞上的包含αvβ3與αvβ5整合素及清道夫受體CD36之受體複合物。膜聯蛋白(Annexin)I屬於Ca2+依賴性磷脂結合蛋白之膜聯蛋白家族,且優先定位於細胞膜之細胞溶質(cytosolic)面上。膜聯蛋白I經顯示係與PS共定位。 The molecular gelin family was subsequently identified through the interaction of its collagen tail with calreticulin (CRT), which in turn was passed through low density lipoprotein (LDL) receptor-related protein (LRP) -1 / CD91) conducts uptake signals (Gardai et al., Cell, 2003; 115: 13-23). As another example, the identified first bridge molecule is thrombospondin (TSP) -1 (Savill et al., J Clin Invest, 1992; 90: 1513-1522), which is used as an extracellular matrix glycoprotein and is considered It binds to the TSP-1 binding site on apoptotic cells and then to the receptor complex on the phagocytes, which contains αvβ3 and αvβ5 integrin and scavenger receptor CD36. Annexin I belongs to the annexin family of Ca2 + -dependent phospholipid binding proteins and is preferentially localized on the cytosolic surface of cell membranes. Annexin I is shown to co-localize with PS.
由RPE對ROS之吞噬對於視網膜功能而言為必需的(Finnemann等人,PNAS,1997;94:12932-937)。據報告參與RPE吞噬ROS之受體包括清道夫受體CD36、整合素受體αvβ5、稱為Mertk之受體酪胺酸激酶、及甘露糖受體(MR)(CD206)(Kevany等人,Physiology,2009;25:8-15)。Finnemann發現,經分離之ROS擁 有經外化的PS,該經外化的PS之阻斷或移除減少ROS於培養物中被RPE結合及吞沒(Finnemann等人,PNAS,2012;109(21):8145-8148)。但是,對於RPE吞噬的理解仍甚少。 The phagocytosis of ROS by RPE is necessary for retinal function (Finnemann et al., PNAS , 1997; 94: 12932-937). Receptors involved in RPE phagocytosis include scavenger receptor CD36, integrin receptor αvβ5, receptor tyrosine kinase called Mertk, and mannose receptor (MR) (CD206) (Kevany et al., Physiology , 2009; 25: 8-15). Finnemann found that the isolated ROS possessed externalized PS, and the blocking or removal of the externalized PS reduced the binding and engulfing of ROS in culture by RPE (Finnemann et al., PNAS, 2012; 109 (21) : 8145-8148). However, there is still little understanding of RPE engulfment.
本發明提供適用於眼科疾病及病症的基於細胞或再生性療法的組成物及方法。詳言之,本發明之特徵在於用於治療眼科疾病或病狀之方法及組成物,包括使用諸如產後衍生細胞之前驅細胞及由該些細胞產生之條件培養基來再生或修復眼組織。產後衍生細胞可為臍帶組織衍生細胞(UTC)或胎盤組織衍生細胞(PDC)。 The present invention provides compositions and methods based on cell-based or regenerative therapies suitable for ophthalmic diseases and disorders. In particular, the invention is characterized by methods and compositions for treating ophthalmic diseases or conditions, including the use of precursor cells such as postpartum-derived cells and conditioned medium produced by these cells to regenerate or repair ocular tissues. The postpartum-derived cells may be umbilical cord tissue-derived cells (UTC) or placental tissue-derived cells (PDC).
本發明之一個態樣為一種治療眼科疾病之方法,其包含向個體投予前驅細胞、或由前驅細胞群製備之條件培養基,其中該等細胞係分泌橋分子。在本發明之一實施例中,橋分子係由條件培養基中之細胞群分泌。在進一步實施例中,橋分子係選自MFG-E8、Gas6、血小板反應蛋白(TSP)-1及TSP-2。在本發明之實施例中,細胞為前驅細胞。在本發明之具體實施例中,細胞為產後衍生細胞。在本發明之實施例中,該等產後衍生細胞係分離自實質上不含血液之人類臍帶組織或胎盤組織。 One aspect of the present invention is a method for treating ophthalmic diseases, which comprises administering precursor cells or a conditioned medium prepared from a population of precursor cells to an individual, wherein the cells secrete bridge molecules. In one embodiment of the invention, the bridge molecule is secreted by the cell population in the conditioned medium. In a further embodiment, the bridge molecule system is selected from MFG-E8, Gas6, thrombospondin (TSP) -1 and TSP-2. In the embodiments of the present invention, the cells are precursor cells. In a specific embodiment of the present invention, the cells are postpartum-derived cells. In an embodiment of the invention, the postpartum-derived cell lines are isolated from human umbilical cord tissue or placental tissue that is substantially free of blood.
在實施例中,前驅細胞(例如產後衍生細胞)群係分泌橋分子。在一實施例中,由前驅細胞(例如產後衍生細胞)群製備之條件培養基含有由該細胞群分泌之橋分子。由該些細胞分泌且分泌於條件培養基中之此類橋分子係選自MFG-E8、Gas6、TSP-1 及TSP-2。產後衍生細胞為臍帶組織衍生細胞(UTC)或胎盤組織衍生細胞(PDC)。 In an embodiment, a population of precursor cells (eg, postpartum-derived cells) secretes bridge molecules. In one embodiment, the conditioned medium prepared from a population of precursor cells (eg, postpartum-derived cells) contains bridge molecules secreted by the population of cells. Such bridge molecules secreted by these cells and secreted in the conditioned medium are selected from MFG-E8, Gas6, TSP-1 and TSP-2. Postpartum-derived cells are umbilical cord tissue-derived cells (UTC) or placental tissue-derived cells (PDC).
在一實施例中,橋分子抑制光受體細胞之細胞凋亡。在另一實施例中,由前驅細胞分泌且分泌於條件培養基中之橋分子減少光受體細胞損失。在一實施例中,光受體細胞損失係藉由橋分子刺激光受體片段之吞噬來減少。 In one embodiment, the bridge molecule inhibits apoptosis of photoreceptor cells. In another embodiment, the bridge molecule secreted by the precursor cells and secreted in the conditioned medium reduces photoreceptor cell loss. In one embodiment, the loss of photoreceptor cells is reduced by phagocytosis of the photoreceptor fragments by bridge molecules.
在另一實施例中,上文所述之細胞群或由上文所述之細胞群製備之條件培養基改質視桿細胞外節(ROS)以利吞噬。在進一步實施例中,橋分子增強視網膜色素上皮(RPE)細胞對ROS之結合及內化。 In another embodiment, the cell population described above or the conditioned medium prepared from the cell population described above modifies the rod outer segment (ROS) to facilitate phagocytosis. In a further embodiment, the bridge molecule enhances the binding and internalization of retinal pigment epithelium (RPE) cells to ROS.
在另一實施例中,上文所述之細胞群或由上文所述之細胞群製備之條件培養基含有由該細胞群分泌之受體酪胺酸激酶(RTK)營養因子。在一特定實施例中,營養因子為BDNF、NT3、HGF、PDGF-CC、PDGF-DD、及GDNF。在實施例中,RTK營養因子媒介視網膜色素上皮(RPE)細胞中之吞噬。 In another embodiment, the cell population described above or the conditioned medium prepared from the cell population described above contains a receptor tyrosine kinase (RTK) trophic factor secreted by the cell population. In a specific embodiment, the nutritional factors are BDNF, NT3, HGF, PDGF-CC, PDGF-DD, and GDNF. In an embodiment, RTK trophic factor mediates phagocytosis in retinal pigment epithelium (RPE) cells.
在某些實施例中,RTK營養因子媒介RPE細胞吞噬以吞噬脫落的光受體片段(由細胞脫落的光受體片段)。在進一步實施例中,RTK營養因子活化RCE細胞上之受體以刺激吞噬。 In certain embodiments, RTK trophic factor-mediated RPE cells phagocytose to engulf shed photoreceptor fragments (photoreceptor fragments shed by cells). In further embodiments, RTK trophic factors activate receptors on RCE cells to stimulate phagocytosis.
本發明之另一態樣之特徵在於一種用於減少視網膜變性中光受體細胞損失之方法,該方法包含向眼睛投予有效減少光受體細胞損失之量的前驅細胞群或由該前驅細胞群製備之條件培養基。在本發明之一實施例中,前驅細胞為產後衍生細胞。在一具體 實施例中,產後衍生細胞係分離自實質上不含血液之人類臍帶組織或胎盤組織。如在其他實施例中,產後衍生細胞係分泌橋分子。在實施例中,條件培養基含有由細胞群(諸如產後衍生細胞群)分泌之橋分子。由產後衍生細胞分泌之此類橋分子係選自MFG-E8、Gas6、TSP-1及TSP-2。 Another aspect of the present invention is characterized by a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering to the eye a precursor cell population or an amount Conditioned medium for group preparation. In one embodiment of the present invention, the precursor cells are postpartum-derived cells. In a specific embodiment, the postpartum-derived cell line is isolated from human umbilical cord tissue or placental tissue that is substantially free of blood. As in other embodiments, the postpartum-derived cell line secretes bridge molecules. In embodiments, the conditioned medium contains bridge molecules secreted by cell populations, such as postpartum-derived cell populations. Such bridge molecules secreted by postpartum-derived cells are selected from MFG-E8, Gas6, TSP-1 and TSP-2.
在另一實施例中,條件培養基係產生自分離的產後衍生細胞或產後衍生細胞群,產後衍生細胞衍生自實質上不含血液之人類臍帶組織或胎盤組織。在實施例中,該產後衍生細胞能夠在培養物中擴增且具有分化成神經表型之細胞的潛能;其中該細胞需要L-纈胺酸以供生長且能夠於至少約5%氧氣中生長。此細胞進一步包含下列特徵中之一或多者:(a)在培養物中進行至少約40次倍增的潛能;(b)在經塗布或未經塗布的組織培養容器上附著並擴增,其中經塗布之組織培養容器包含明膠、層黏蛋白、膠原蛋白、聚鳥胺酸、玻璃連接蛋白、或纖連蛋白素之塗層;(c)生產組織因子、波形蛋白(vimentin)、及α-平滑肌肌動蛋白(alpha-smooth muscle actin)中之至少一者;(d)生產CD10、CD13、CD44、CD73、CD90、PDGFr-α、PD-L2及HLA-A、HLA-B、HLA-C中之至少一者;(e)不生產CD31、CD34、CD45、CD80、CD86、CD117、CD141、CD178、B7-H2、HLA-G、及HLA-DR、HLA-DP、HLA-DQ中之至少一者,如藉由流動式細胞測量術所偵測;(f)針對編碼下列之至少一種基因的基因表現(相對於纖維母細胞、間葉幹細胞或髂骨崤骨髓細胞之人類細胞)係經增加:介白素8;內質網蛋白(reticulon)1;趨化激素(C--X--C 模體)配體1(黑色素瘤生長刺激活性物,α);趨化激素(C--X--C模體)配體6(顆粒性細胞趨化蛋白質2);趨化激素(C--X--C模體)配體3;腫瘤壞死因子,α-誘發蛋白質3;C型凝集素超家族成員2;威爾姆斯(Wilms)腫瘤1;醛去氫酶1家族成員A2;腎素(renin);氧化低密度脂蛋白受體1;智人(Homo sapiens)殖株IMAGE:4179671;蛋白質激酶C ζ;假想蛋白質DKFZp564F013;卵巢癌向下調控因子1;及殖株DKFZp547k1113之智人基因;(g)針對編碼下列之至少一種基因的基因表現(相對於纖維母細胞、間葉幹細胞或髂骨崤骨髓細胞之人類細胞)係經減少:矮小同源盒(short stature homeobox)2;熱休克27kDa蛋白質2;趨化激素(C--X--C模體)配體12(基質細胞衍生因子1);彈性蛋白(瓣上主動脈狹窄,威廉-波倫(Williams-Beuren)症候群);智人(Homo sapiens)mRNA;cDNA DKFZp586M2022(來自殖株DKFZp586M2022);間葉同源盒2(生長停滯特異性同源盒(growth arrest-specific homeo box));sine oculis同源盒同源物1(果蠅(Drosophila));αB水晶體蛋白;形態發生紊亂關聯活化物2(disheveled associated activator of morphogenesis 2);DKFZP586B2420蛋白質;類似於neuralin 1者;結合四素(tetranectin,纖維蛋白溶酶原(plasminogen)結合蛋白質);src同源體3(SH3)與多半胱胺酸區(cysteine rich domain);膽固醇25-羥化酶;矮小相關轉錄因子3(runt-related transcription factor 3);介白素11受體,α;原膠原蛋白C-內肽酶增強子(procollagen C-endopeptidase enhancer);捲曲同源物7(frizzled homolog 7)(果 蠅);假想基因BC008967;第三型膠原蛋白α1;固生蛋白C(tenascin C,hexabrachion);易洛魁族同源盒蛋白質5(iroquois homeobox protein 5);希菲斯特蛋白(hephaestin);整合素β8;突觸囊泡醣蛋白(synaptic vesicle glycoprotein)2;神經胚細胞瘤,致腫瘤性抑制因子1;類胰島素生長因子結合蛋白質2,36kDa;智人cDNA FLJ12280 fis,殖株MAMMA1001744;類細胞介素受體因子1;鉀中間物/小電導鈣活化通道,次家族N,成員4;整合素β7;具有PDZ-結合模體之轉錄共活化物(TAZ);sine oculis同源盒同源物2(果蠅);KIAA1034蛋白質;囊泡相關膜蛋白質5(肌短蛋白(myobrevin));含EGF類fibulin細胞外基質蛋白質(EGF-containing fibulin-like extracellular matrix protein)1;早期生長反應蛋白質(early growth response)3;無背端同源盒(distal-less homeo box)5;假想蛋白質FLJ20373;醛基-酮基還原酶家族1,成員C3(3-α羥類固醇去氫酶(3-alpha hydroxysteroid dehydrogenase),II型);雙聚醣(biglycan);具有PDZ-結合模體之轉錄共活化物(TAZ);纖連蛋白素1;腦啡肽前質(proenkephalin);整合素,類β1(具有類EGF重複區);智人mRNA全長插入物cDNA殖株EUROIMAGE 1968422;EphA3;KIAA0367蛋白質;利尿鈉胜肽受體C(natriuretic peptide receptor C)/鳥苷酸環化酶C(guanylate cyclase C)(心房利尿鈉胜肽受體C(atrionatriuretic peptide receptor C));假想蛋白質FLJ14054;智人(Homo sapiens)mRNA;cDNA DKFZp564B222(來自殖株DKFZp564B222);BCL2/類腺病毒E1B 19kDa交互作用蛋白質3 (adenovirus E1B 19kDa interacting protein 3-like);AE結合蛋白質1;細胞色素c氧化酶次單元VIIa多肽1(肌肉);類似於neuralin 1者;B細胞轉位基因1;假想蛋白質FLJ23191;及DKFZp586L151;及(h)不表現hTERT或端粒酶。在一實施例中,臍帶組織衍生細胞進一步具有以下特徵:(i)分泌MCP-1、IL-6、IL-8、GCP-2、HGF、KGF、FGF、HB-EGF、BDNF、TPO、MIP1b、I309、MDC、RANTES、及TIMP1中之至少一者;(j)不分泌TGF-β2、MIP1a、ANG2、PDGFbb、及VEGF中之至少一者,如藉由ELISA所偵測。在另一實施例中,胎盤組織衍生細胞進一步具有以下特徵:(i)分泌MCP-1、IL-6、IL-8、GCP-2、HGF、KGF、HB-EGF、BDNF、TPO、MIP1a、RANTES、及TIMP1中之至少一者;(j)不分泌TGF-β2、MIP1b、ANG2、PDGFbb、FGF、及VEGF中之至少一者,如藉由ELISA所偵測。 In another embodiment, the conditioned medium line is produced from isolated postpartum-derived cells or a population of postpartum-derived cells derived from human umbilical cord tissue or placental tissue that is substantially free of blood. In an embodiment, the postpartum-derived cells are capable of expanding in culture and have the potential to differentiate into neuronal phenotype cells; wherein the cells require L-valine for growth and can grow in at least about 5% oxygen . This cell further includes one or more of the following characteristics: (a) the potential to perform at least about 40 doublings in culture; (b) attachment and expansion on coated or uncoated tissue culture vessels, wherein The coated tissue culture container contains a coating of gelatin, laminin, collagen, polyguanylic acid, glass connexin, or fibronectin; (c) production of tissue factor, vimentin, and α- At least one of alpha-smooth muscle actin; (d) Production of CD10, CD13, CD44, CD73, CD90, PDGFr-α, PD-L2 and HLA-A, HLA-B, HLA-C At least one of; (e) does not produce at least one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR, HLA-DP, HLA-DQ One, as detected by flow cytometry; (f) for the expression of genes encoding at least one of the following genes (relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells). Increase: Interleukin 8; Reticulon 1; Chemokine (C--X--C motif) ligand 1 (melanoma growth stimulation) Sex, α); chemokine (C--X--C motif) ligand 6 (granulocyte chemoattractant protein 2); chemokine (C--X--C motif) ligand 3 ; Tumor necrosis factor, alpha-evoked protein 3; C-type lectin superfamily member 2; Wilms tumor 1; aldehyde dehydrogenase 1 family member A2; renin; oxidized low-density lipoprotein Receptor 1; Homo sapiens strain IMAGE: 4179671; protein kinase C ζ; hypothetical protein DKFZp564F013; ovarian cancer down-regulatory factor 1; and the strain Homo sapiens gene DKFZp547k1113; The gene expression of a gene (human cells relative to fibroblasts, mesenchymal stem cells, or iliac bone marrow cells) is reduced: short stature homeobox 2; heat shock 27kDa protein 2; chemokine ( C--X--C motif) ligand 12 (stromal cell-derived factor 1); elastin (supervalvular aortic stenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (from strain DKFZp586M2022); mesenchymal homeobox 2 (growth arrest-specific homeo box )); Sine oculis homology box homolog 1 (Drosophila); αB aquatic protein; morphogenesis disorder associated activator 2 (disheveled associated activator of morphogenesis 2); DKFZP586B2420 protein; similar to neuralin 1; binding Tetranectin (plasminogen binding protein); src homolog 3 (SH3) and polycysteine rich domain (cysteine rich domain); cholesterol 25-hydroxylase; dwarf related transcription factor 3 ( runt-related transcription factor 3); interleukin 11 receptor, α; procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila); Hypothetical gene BC008967; type III collagen α1; tenascin C (tenascin C, hexabrachion); iroquois homeobox protein 5 (iroquois homeobox protein 5); hephaestin (hephaestin); integrin β8 ; Synaptic vesicle glycoprotein 2; Neuroblastoma, tumorigenic inhibitory factor 1; Insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNA FLJ12280 fis, strain MAMMA10017 44; Interleukin-like receptor factor 1; potassium intermediate / small conductivity calcium activated channel, subfamily N, member 4; integrin β7; transcription coactivator (TAZ) with PDZ-binding motif; sine oculis Source box homolog 2 (Drosophila); KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin); EGF-containing fibulin-like extracellular matrix protein 1 (EGF-containing fibulin-like extracellular matrix protein) 1; Early growth response protein 3; distant-less homeo box 5; hypothetical protein FLJ20373; aldehyde-keto reductase family 1, member C3 (3-α-hydroxysteroid dehydrogenation) Enzyme (3-alpha hydroxysteroid dehydrogenase), type II); biglycan; transcription coactivator (TAZ) with PDZ-binding motif; fibronectin 1; proenkephalin; Integrin, β1-like (with EGF-like repeat region); Homo sapiens mRNA full-length insert cDNA strain EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptor C (natriuretic peptide receptor C) / guanylate cyclase C (guanylate cyclase C) (atrial natriuretic peptide receptor C (a trionatriuretic peptide receptor C)); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222 (from the strain DKFZp564B222); BCL2 / adenovirus E1B 19kDa interacting protein 3 (adenovirus E1B 19kDa interacting protein 3-like); AE binding protein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle); similar to neuralin 1; B cell translocation gene 1; hypothetical protein FLJ23191; and DKFZp586L151; and (h) does not express hTERT or telomerase . In one embodiment, umbilical cord tissue-derived cells further have the following characteristics: (i) secretion of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b , I309, MDC, RANTES, and TIMP1; (j) does not secrete at least one of TGF-β2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA. In another embodiment, the placental tissue-derived cells further have the following characteristics: (i) secretion of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, HB-EGF, BDNF, TPO, MIP1a, At least one of RANTES and TIMP1; (j) does not secrete at least one of TGF-β2, MIP1b, ANG2, PDGFbb, FGF, and VEGF, as detected by ELISA.
在特定實施例中,產後衍生細胞具有以下細胞型之所有識別特徵:細胞型UMB 022803(P7)(ATCC存取號PTA-6067);細胞型UMB 022803(P17)(ATCC存取號PTA-6068)、細胞型PLA 071003(P8)(ATCC存取號PTA-6074);細胞型PLA 071003(P11)(ATCC存取號PTA-6075);或細胞型PLA 071003(P16)(ATCC存取號PTA-6079)。在一實施例中,衍生自臍組織之產後衍生細胞具有細胞型UMB 022803(P7)(ATCC存取號PTA-6067)或細胞型UMB 022803(P17)(ATCC存取號PTA-6068)之所有識別特徵。在另一實施例中,衍生自胎盤組織之產後衍生細胞具有以下細胞型之所有 識別特徵:細胞型PLA 071003(P8)(ATCC存取號PTA-6074);細胞型PLA 071003(P11)(ATCC存取號PTA-6075);或細胞型PLA 071003(P16)(ATCC存取號PTA-6079)。 In a particular embodiment, the postpartum-derived cells have all the identifying characteristics of the following cell types: cell type UMB 022803 (P7) (ATCC access number PTA-6067); cell type UMB 022803 (P17) (ATCC access number PTA-6068 ), Cell type PLA 071003 (P8) (ATCC access number PTA-6074); cell type PLA 071003 (P11) (ATCC access number PTA-6075); or cell type PLA 071003 (P16) (ATCC access number PTA -6079). In one embodiment, the postpartum-derived cells derived from umbilical tissue have all of the cell type UMB 022803 (P7) (ATCC access number PTA-6067) or cell type UMB 022803 (P17) (ATCC access number PTA-6068) Identify features. In another embodiment, postpartum-derived cells derived from placental tissue have all the identifying characteristics of the following cell types: cell type PLA 071003 (P8) (ATCC accession number PTA-6074); cell type PLA 071003 (P11) (ATCC Accession number PTA-6075); or cell type PLA 071003 (P16) (ATCC accession number PTA-6079).
在某些實施例中,產後衍生細胞在一或多種酶活性存在下分離,該些酶活性包含金屬蛋白酶活性、黏液分解活性及中性蛋白酶活性。較佳地,產後衍生細胞具有正常核型,其在細胞繼代培養時維持。在較佳實施例中,產後衍生細胞表現CD10、CD13、CD44、CD73、CD90中之各者。在實施例中,產後衍生細胞表現CD10、CD13、CD44、CD73、CD90、PDGFr-α、及HLA-A、B、C中之各者。在較佳實施例中,產後衍生細胞不表現CD31、CD34、CD45、CD117。在實施例中,產後衍生細胞不表現CD31、CD34、CD45、CD117、CD141、或HLA-DR、DP、DQ,如藉由流動式細胞測量術所偵測。在實施例中,該些細胞不表現hTERT或端粒酶。 In certain embodiments, the postpartum-derived cells are isolated in the presence of one or more enzyme activities, including metalloproteinase activity, mucus decomposition activity, and neutral protease activity. Preferably, the postpartum-derived cells have a normal karyotype, which is maintained during the subculture of the cells. In a preferred embodiment, the postpartum-derived cells express each of CD10, CD13, CD44, CD73, and CD90. In the examples, the postpartum-derived cells express each of CD10, CD13, CD44, CD73, CD90, PDGFr-α, and HLA-A, B, and C. In a preferred embodiment, the postpartum-derived cells do not express CD31, CD34, CD45, CD117. In an embodiment, the postpartum-derived cells do not express CD31, CD34, CD45, CD117, CD141, or HLA-DR, DP, DQ, as detected by flow cytometry. In an embodiment, these cells do not express hTERT or telomerase.
在如上文之實施例中,細胞群為實質上均質性產後衍生細胞群。在特定實施例中,該族群為均質性產後衍生細胞群。在本發明之實施例中,產後衍生細胞係衍生自實質上不含血液之人類臍帶組織或胎盤組織。 In the embodiment as above, the cell population is a substantially homogeneous postpartum-derived cell population. In certain embodiments, the population is a homogeneous postpartum-derived cell population. In an embodiment of the invention, the postpartum-derived cell line is derived from human umbilical cord tissue or placental tissue that is substantially free of blood.
在某些實施例中,如上文所述之產後衍生細胞群或由該細胞群製備之條件培養基係與至少一種其他細胞型一起投予,該等其他細胞型諸如星狀細胞、寡樹突細胞、神經元、神經前驅、神經幹細胞、視網膜上皮幹細胞、角膜上皮幹細胞、或其他多潛能性或多能性幹細胞。在該些實施例中,其他細胞型可與產後衍生細胞 群或由產後衍生細胞群製備之條件培養基同時、在之前、或在之後投予。 In certain embodiments, the postpartum-derived cell population as described above or a conditioned medium prepared from the cell population is administered with at least one other cell type, such as stellate cells, oligodendrocyte , Neurons, neural precursors, neural stem cells, retinal epithelial stem cells, corneal epithelial stem cells, or other pluripotent or pluripotent stem cells. In these embodiments, other cell types may be administered simultaneously, before, or after the postpartum-derived cell population or the conditioned medium prepared from the postpartum-derived cell population.
同樣地,在這些及其他實施例中,如上文所述之產後衍生細胞群或由該產後衍生細胞群製備之條件培養基係與至少一種其他藥劑諸如用於眼治療之藥品、或另一有益的輔助劑諸如消炎劑、抗細胞凋亡劑、抗氧化劑或生長因子一起投予。在該些實施例中,其他藥劑可與產後衍生細胞群或由產後衍生細胞群製備之條件培養基同時、在之前、或在之後投予。 Similarly, in these and other embodiments, the postpartum-derived cell population or the conditioned medium prepared from the postpartum-derived cell population as described above is combined with at least one other agent such as a drug for eye treatment, or another beneficial Adjuvants such as anti-inflammatory agents, anti-apoptotic agents, antioxidants or growth factors are administered together. In these embodiments, other agents may be administered simultaneously, before, or after the postpartum-derived cell population or the conditioned medium prepared from the postpartum-derived cell population.
在本文所述之各種實施例中,產後衍生細胞群(臍或胎盤)或由產後衍生細胞產生之條件培養基係向眼睛例如眼睛表面投予、或向眼睛內部或向靠近眼睛之位置例如於眼睛後面投予。產後衍生細胞群或由該產後衍生細胞群製備之條件培養基可透過套管或自植入於眼睛內或靠近眼睛之患者體內的裝置投予,或可藉由植入具有細胞群或條件培養基之基質或支架來投予。 In the various embodiments described herein, the postpartum-derived cell population (umbilical or placenta) or conditioned medium produced by the postpartum-derived cells is administered to the eye, such as the surface of the eye, or to the interior of the eye or to a location near the eye, such as Give later. The postpartum-derived cell population or the conditioned medium prepared from the postpartum-derived cell population can be administered through a cannula or from a device implanted in or near a patient in the eye, or can be implanted with a cell population or conditioned medium Substrate or scaffold to administer.
本發明之另一態樣之特徵在於一種用於減少視網膜變性病狀中光受體細胞損失之組成物,該組成物包含有效減少光受體細胞損失之量的前驅細胞群或由前驅細胞群製備之條件培養基。較佳地,前驅細胞為如上文所述之產後衍生細胞。更佳地,產後衍生細胞係分離自如上文所述之實質上不含血液之產後臍帶或胎盤。變性病狀可為急性、慢性或進行性病狀。 Another aspect of the present invention is characterized by a composition for reducing the loss of photoreceptor cells in a retinal degeneration pathology, the composition comprising a precursor cell population or a precursor cell population effective to reduce the amount of photoreceptor cell loss Prepared conditioned medium. Preferably, the precursor cells are post-natally derived cells as described above. More preferably, the postpartum-derived cell line is isolated from a postpartum umbilical cord or placenta that is substantially free of blood as described above. A degenerative condition can be acute, chronic, or progressive.
在某些實施例中,如上文所述之組成物包含至少一種其他細胞型,諸如星狀細胞、寡樹突細胞、神經元、神經前驅、神 經幹細胞、視網膜上皮幹細胞、角膜上皮幹細胞、或其他多潛能性或多能性幹細胞。在這些或其他實施例中,組成物包含至少一種其他藥劑,諸如用於治療眼變性病症之藥品或其他有益的輔助試劑,例如消炎劑、抗細胞凋亡劑、抗氧化劑或生長因子。 In certain embodiments, the composition as described above includes at least one other cell type, such as stellate cells, oligodendritic cells, neurons, neural precursors, neural stem cells, retinal epithelial stem cells, corneal epithelial stem cells, or other Pluripotent or pluripotent stem cells. In these or other embodiments, the composition contains at least one other medicament, such as a medicament for treating an ocular degenerative disorder or other beneficial auxiliary agents, such as anti-inflammatory agents, anti-apoptotic agents, antioxidants or growth factors.
在如上文所述之實施例中,組成物係進一步包含醫藥上可接受之載劑之醫藥組成物。在某些實施例中,醫藥組成物經調配用於向眼睛表面投予。或者,其可經調配用於向眼睛內部或靠近眼睛處(例如眼睛後面)投予。醫藥組成物亦可調配為含有如上文所述之前驅細胞或由前驅細胞製備之條件培養基之基質或支架。 In the embodiments as described above, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is formulated for administration to the surface of the eye. Alternatively, it can be formulated for administration into or near the eye (eg, behind the eye). The pharmaceutical composition can also be formulated as a matrix or scaffold containing precursor cells as described above or a conditioned medium prepared from the precursor cells.
根據本發明之另一態樣,提供一種用於治療具有眼變性病狀之患者的套組。該套組包含醫藥上可接受之載劑、前驅細胞或由前驅細胞(諸如分離自產後組織之細胞,較佳為上文所述之產後衍生細胞)產生之條件培養基、及於治療患者之方法中使用該套組之說明書。該套組亦可含有一或多種額外組分,諸如用於產生條件培養基之試劑及說明書、或至少一種其他細胞型之族群、或一或多種可用於治療眼變性病狀之劑。 According to another aspect of the present invention, there is provided a kit for treating patients with ocular degeneration. The kit contains a pharmaceutically acceptable carrier, precursor cells or conditioned medium produced by the precursor cells (such as cells isolated from postpartum tissues, preferably the postpartum derived cells described above), and methods for treating patients Use the instruction manual of the set. The kit may also contain one or more additional components, such as reagents and instructions for producing conditioned medium, or at least one other cell type population, or one or more agents that can be used to treat ocular degeneration.
本發明之其他態樣包括一種減少視網膜變性中光受體細胞損失之方法,該方法包含投予有效減少光受體細胞損失之量的包含前驅細胞群或由前驅細胞群製備之條件培養基的組成物。較佳地,前驅細胞為產後衍生細胞或條件培養基係由如本文所述之產後衍生細胞群製備。在本發明之實施例中,產後衍生細胞係分離自實質上不含血液之臍帶組織或胎盤組織。在特定實施例中,產後衍 生細胞或由產後衍生細胞群製備之條件培養基含有由該細胞群分泌之橋分子。由該等產後衍生細胞分泌或分泌於條件培養基中之此類橋分子係選自MFG-E8、Gas6、TSP-1、及TSP-2。 Other aspects of the invention include a method of reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering a composition of conditioned medium comprising or prepared from a precursor cell population in an amount effective to reduce the loss of photoreceptor cells Thing. Preferably, the precursor cells are postpartum-derived cells or the conditioned medium is prepared from the postpartum-derived cell population as described herein. In an embodiment of the invention, the postpartum-derived cell line is isolated from umbilical cord tissue or placental tissue that is substantially free of blood. In certain embodiments, the postpartum-derived cells or conditioned medium prepared from the postpartum-derived cell population contains bridge molecules secreted by the cell population. Such bridge molecules secreted by the postpartum-derived cells or secreted in the conditioned medium are selected from MFG-E8, Gas6, TSP-1, and TSP-2.
在一些實施例中,本發明提供一種用於減少視網膜變性中光受體細胞損失之方法,該方法包含向眼睛投予有效減少或預防光受體細胞損失之量的產後衍生細胞或由產後衍生細胞群製備之條件培養基。產後衍生細胞係衍生自實質上不含血液之臍帶組織或胎盤組織。在一些實施例中,產後衍生細胞群為實質上均質性族群。在具體實施例中,細胞群為均質性。 In some embodiments, the present invention provides a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering to the eye a postpartum-derived cell in an amount effective to reduce or prevent loss of photoreceptor cells or derived from postpartum Conditioned medium prepared from cell populations. The postpartum-derived cell line is derived from umbilical cord tissue or placental tissue that is substantially free of blood. In some embodiments, the postpartum-derived cell population is a substantially homogeneous population. In a specific embodiment, the cell population is homogeneous.
在本文所述之本發明之進一步態樣中,產後衍生細胞群(臍或胎盤)或由產後衍生細胞產生之條件培養基保護視網膜細胞或改善因氧化應力或氧化損傷所引起的視網膜損傷。在一實施例中,本發明為一種減少視網膜變性之方法,該方法包含向眼睛投予有效減少或防止氧化應力或損傷之量的產後衍生細胞群或由產後衍生細胞群產生之條件培養基。在本發明之實施例中,產後衍生細胞係分離自實質上不含血液之臍帶組織或胎盤組織。在實施例中,視網膜細胞及組織暴露至氧化應力或氧化損傷。在本文之實施例中,視網膜細胞及組織為光受體細胞或視網膜上皮,包括視網膜色素上皮(RPE)細胞。在本文之實施例中,氧化應力或氧化損傷為高氧張力、日光暴露,包括長期日光暴露。 In a further aspect of the invention described herein, the postpartum-derived cell population (umbilical or placenta) or conditioned medium produced by the postpartum-derived cells protects retinal cells or improves retinal damage caused by oxidative stress or oxidative damage. In one embodiment, the invention is a method of reducing retinal degeneration, the method comprising administering to the eye a postpartum-derived cell population or a conditioned medium produced from the postpartum-derived cell population in an amount effective to reduce or prevent oxidative stress or damage. In an embodiment of the invention, the postpartum-derived cell line is isolated from umbilical cord tissue or placental tissue that is substantially free of blood. In an embodiment, retinal cells and tissues are exposed to oxidative stress or oxidative damage. In the embodiments herein, the retinal cells and tissues are photoreceptor cells or retinal epithelium, including retinal pigment epithelium (RPE) cells. In the embodiments herein, the oxidative stress or oxidative damage is high oxygen tension, sun exposure, including long-term sun exposure.
一實施例為一種減少視網膜變性中光受體細胞損失之方法,該方法包含向眼睛投予有效減少或防止氧化應力或損傷之 量的產後衍生細胞群或由產後衍生細胞群產生之條件培養基。在實施例中,產後衍生細胞係分離自實質上不含血液之臍帶組織或胎盤組織。在實施例中,視網膜細胞及組織暴露至氧化應力或氧化損傷。在本文之實施例中,視網膜細胞及組織為光受體細胞或視網膜上皮,包括視網膜色素上皮(RPE)細胞。在本文之實施例中,氧化應力或氧化損傷係選自高氧張力、日光暴露(包括長期日光暴露)、自由基應力、光氧化、及光誘導損傷。 One embodiment is a method of reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering to the eye a postpartum-derived cell population or a conditioned medium produced from the postpartum-derived cell population in an amount effective to reduce or prevent oxidative stress or damage. In an embodiment, the postpartum-derived cell line is isolated from umbilical cord tissue or placental tissue that is substantially free of blood. In an embodiment, retinal cells and tissues are exposed to oxidative stress or oxidative damage. In the embodiments herein, the retinal cells and tissues are photoreceptor cells or retinal epithelium, including retinal pigment epithelium (RPE) cells. In the embodiments herein, the oxidative stress or oxidative damage is selected from high oxygen tension, solar exposure (including long-term solar exposure), free radical stress, photo-oxidation, and photo-induced damage.
在一實施例中,本發明為一種用於減少視網膜變性中光受體細胞損失之方法,該方法包含投予有效減少或預防光受體細胞損失之量的產後衍生細胞群或由產後衍生細胞群產生之條件培養基,其中該細胞群係分離自實質上不含血液之產後組織,且其中該細胞群能夠在培養物中擴增、具有分化成至少一神經表型之細胞的潛能、在繼代之後維持正常核型、且具有以下特徵:a)在培養物中進行40次族群倍增的潛能;b)生產CD10、CD13、CD44、CD73、及CD90;及c)不生產CD31、CD34、CD45、CD117、及CD141,且其中產後衍生細胞群係分泌橋分子,其中由產後衍生細胞群製備之條件培養基含有由該細胞群分泌之橋分子。在一些實施例中,由產後衍生細胞分泌之橋分子係選自MFG-E8、Gas6、TSP-1及TSP-2。 在一些實施例中,細胞群為實質上均質性族群。在具體實施例中,細胞群為均質性。產後衍生細胞為臍帶組織衍生細胞或胎盤組織衍生細胞。在一實施例中,臍帶組織衍生細胞群分泌MCP-1、IL-6、 IL-8、GCP-2、HGF、KGF、FGF、HB-EGF、BDNF、TPO、MIP1b、I309、MDC、RANTES、及TIMP1。在實施例中,臍帶組織衍生細胞群不分泌TGF-β2、MIP1a、ANG2、PDGFbb、及VEGF,如藉由ELISA所偵測。在另一實施例中,胎盤組織衍生細胞群分泌MCP-1、IL-6、IL-8、GCP-2、HGF、KGF、HB-EGF、BDNF、TPO、MIP1a、RANTES、及TIMP1。在實施例中,胎盤組織衍生細胞群不分泌TGF-β2、MIP1b、ANG2、PDGFbb、FGF、及VEGF,如藉由ELISA所偵測。在實施例中,臍衍生細胞或胎盤衍生細胞為對HLA-A、HLA-B、HLA-C陽性,及對HLA-DR、HLA-DP、HLA-DQ陰性。在進一步實施例中,臍衍生細胞不表現hTERT或端粒酶。 In one embodiment, the invention is a method for reducing the loss of photoreceptor cells in retinal degeneration, the method comprising administering a postpartum-derived cell population or cells derived from postpartum in an amount effective to reduce or prevent the loss of photoreceptor cells A conditioned medium produced by a population, wherein the cell population is isolated from a postpartum tissue that is substantially free of blood, and wherein the population of cells can be expanded in culture, has the potential to differentiate into cells with at least one neurophenotype, Maintain normal karyotype after generation and have the following characteristics: a) Potential for 40 population doublings in culture; b) Production of CD10, CD13, CD44, CD73, and CD90; and c) No production of CD31, CD34, CD45 , CD117, and CD141, and the postpartum-derived cell population secretes bridge molecules, and the conditioned medium prepared from the postpartum-derived cell population contains bridge molecules secreted by the cell population. In some embodiments, the bridge molecule secreted by the postpartum-derived cells is selected from MFG-E8, Gas6, TSP-1, and TSP-2. In some embodiments, the cell population is a substantially homogeneous population. In a specific embodiment, the cell population is homogeneous. Postpartum-derived cells are umbilical cord tissue-derived cells or placental tissue-derived cells. In one embodiment, the umbilical cord tissue-derived cell population secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, I309, MDC, RANTES, And TIMP1. In an embodiment, the umbilical cord tissue-derived cell population does not secrete TGF-β2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA. In another embodiment, the placental tissue-derived cell population secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1. In an embodiment, the placental tissue-derived cell population does not secrete TGF-β2, MIP1b, ANG2, PDGFbb, FGF, and VEGF, as detected by ELISA. In the embodiment, the umbilical-derived cells or placenta-derived cells are positive for HLA-A, HLA-B, HLA-C, and negative for HLA-DR, HLA-DP, HLA-DQ. In further embodiments, umbilical derived cells do not express hTERT or telomerase.
在一實施例中,本發明為一種用於減少視網膜變性中光受體細胞損失之方法,該方法包含投予有效減少光受體細胞損失之量的產後衍生細胞群或由產後衍生細胞群製備之條件培養基,其中該細胞群係分離自實質上不含血液之人類臍帶組織,且其中該細胞群能夠在培養物中擴增、具有分化成至少一神經表型之細胞的潛能、在繼代之後維持正常核型、且具有以下特徵:a)在培養物中進行40次族群倍增的潛能;b)生產CD10、CD13、CD44、CD73、及CD90;c)不生產CD31、CD34、CD45、CD117、及CD141,以及d)相對於纖維母細胞、間葉幹細胞、或髂骨崤骨髓細胞之人類細胞,增加編碼介白素8及內質網蛋白1之基因的表現,且 其中產後衍生細胞群係分泌橋分子,或其中由產後衍生細胞群製備之條件培養基含有由該細胞群分泌之橋分子。在實施例中,由產後衍生細胞群分泌之橋分子、或由產後衍生細胞群所分泌之條件培養基中之橋分子係選自MFG-E8、Gas6、TSP-1及TSP-2。在實施例中,細胞群分泌MCP-1、IL-6、IL-8、GCP-2、HGF、KGF、FGF、HB-EGF、BDNF、TPO、MIP1b、I309、MDC、RANTES、及TIMP1。在一些實施例中,細胞群不分泌TGF-β2、MIP1a、ANG2、PDGFbb、及VEGF,如藉由ELISA所偵測。在實施例中,細胞群為對HLA-A、HLA-B、HLA-C陽性,及對HLA-DR、HLA-DP、HLA-DQ陰性。在一些實施例中,細胞群為實質上均質性族群。在具體實施例中,細胞群為均質性。另外,細胞群不表現hTERT或端粒酶。 In one embodiment, the present invention is a method for reducing photoreceptor cell loss in retinal degeneration, the method comprising administering or prepared from a postpartum-derived cell population effective to reduce the amount of photoreceptor cell loss Conditioned medium, wherein the cell population is isolated from human umbilical cord tissue that is substantially free of blood, and wherein the cell population can be expanded in culture, has the potential to differentiate into cells with at least one neurophenotype, and is passaged The normal karyotype is maintained afterwards and has the following characteristics: a) the potential for 40 population doublings in culture; b) production of CD10, CD13, CD44, CD73, and CD90; c) no production of CD31, CD34, CD45, CD117 , And CD141, and d) Compared with human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells, the expression of genes encoding interleukin 8 and endoplasmic reticulum protein 1 is increased, and the post-natal derived cell population Is a secreted bridge molecule, or a conditioned medium prepared from a postpartum-derived cell population contains a bridge molecule secreted by the cell population. In an embodiment, the bridge molecule secreted by the postpartum-derived cell population or the bridge molecule in the conditioned medium secreted by the postpartum-derived cell population is selected from MFG-E8, Gas6, TSP-1, and TSP-2. In an embodiment, the cell population secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, I309, MDC, RANTES, and TIMP1. In some embodiments, the cell population does not secrete TGF-β2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA. In the examples, the cell population is positive for HLA-A, HLA-B, HLA-C, and negative for HLA-DR, HLA-DP, HLA-DQ. In some embodiments, the cell population is a substantially homogeneous population. In a specific embodiment, the cell population is homogeneous. In addition, the cell population did not express hTERT or telomerase.
在某些實施例中,本發明提供一種用於減少視網膜變性中光受體細胞損失之方法,該方法包含投予有效減少或預防光受體細胞損失之量的臍衍生細胞群或由臍衍生細胞群製備之條件培養基,其中該細胞群係分離自實質上不含血液之人類臍帶組織,且其中該細胞群能夠在培養物中擴增、具有分化成至少一神經表型之細胞的潛能、且具有以下特徵:a. 在培養物中進行40次族群倍增的潛能;b. 生產CD10、CD13、CD44、CD73、及CD90;及c. 相對於纖維母細胞、間葉幹細胞、或髂骨崤骨髓細胞之人類細胞,增加編碼介白素8及內質網蛋白1之基因的表現,且 產後衍生細胞群係分泌橋分子,其中由產後衍生細胞群製備之條件培養基含有由該細胞群分泌之橋分子。在實施例中,在分泌橋分子之產後衍生細胞群中分泌之橋分子、或分泌於條件培養基中之橋分子係選自MFG-E8、Gas6、TSP-1及TSP-2。在實施例中,該些細胞不生產或為對CD31、CD34、CD45、CD117、及CD141陰性。在一實施例中,臍帶組織衍生細胞群分泌MCP-1、IL-6、IL-8、GCP-2、HGF、KGF、FGF、HB-EGF、BDNF、TPO、MIP1b、I309、MDC、RANTES、及TIMP1,且不分泌TGF-β2、MIP1a、ANG2、PDGFbb、及VEGF,如藉由ELISA所偵測。在實施例中,臍衍生細胞群為對HLA-A、HLA-B、HLA-C陽性,及對HLA-DR、HLA-DP、HLA-DQ陰性。另外,細胞群不表現hTERT或端粒酶。在一些實施例中,細胞群為實質上均質性族群。在具體實施例中,細胞群為均質性。 In certain embodiments, the present invention provides a method for reducing photoreceptor cell loss in retinal degeneration, the method comprising administering an umbilical-derived cell population in an amount effective to reduce or prevent photoreceptor cell loss or derived from the umbilical cord A conditioned medium prepared by a cell population, wherein the cell population is isolated from human umbilical cord tissue that is substantially free of blood, and wherein the cell population can be expanded in culture and has the potential to differentiate into cells with at least one neurophenotype, And has the following characteristics: a. Potential for 40 times population doubling in culture; b. Production of CD10, CD13, CD44, CD73, and CD90; and c. Relative to fibroblasts, mesenchymal stem cells, or iliac bones Human cells of bone marrow cells increase the expression of genes encoding interleukin 8 and endoplasmic reticulum protein 1, and the postpartum-derived cell population secretes bridge molecules, in which the conditioned medium prepared from the postpartum-derived cell population contains the Bridge molecule. In an embodiment, the bridge molecule secreted in the postpartum-derived cell population secreting the bridge molecule, or the bridge molecule secreted in the conditioned medium is selected from MFG-E8, Gas6, TSP-1, and TSP-2. In the examples, these cells are not produced or are negative for CD31, CD34, CD45, CD117, and CD141. In one embodiment, the umbilical cord tissue-derived cell population secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, I309, MDC, RANTES, And TIMP1, and does not secrete TGF-β2, MIP1a, ANG2, PDGFbb, and VEGF, as detected by ELISA. In the examples, the umbilical-derived cell population is positive for HLA-A, HLA-B, HLA-C, and negative for HLA-DR, HLA-DP, HLA-DQ. In addition, the cell population did not express hTERT or telomerase. In some embodiments, the cell population is a substantially homogeneous population. In a specific embodiment, the cell population is homogeneous.
本發明之另一態樣為一種製作條件培養基之方法,該方法包含培養細胞群,其中該條件培養基含有由該細胞群分泌之橋分子。在本發明之一實施例中,橋分子係由條件培養基中之細胞群分泌。在進一步實施例中,橋分子係選自MFG-E8、Gas6、血小板反應蛋白(TSP)-1及TSP-2。在本發明之實施例中,細胞為前驅細胞。在本發明之具體實施例中,細胞為產後衍生細胞。在本發明之實施例中,該等產後衍生細胞係分離自實質上不含血液之人類臍帶組織或胎盤組織。 Another aspect of the invention is a method of making a conditioned medium, the method comprising culturing a cell population, wherein the conditioned medium contains bridge molecules secreted by the cell population. In one embodiment of the invention, the bridge molecule is secreted by the cell population in the conditioned medium. In a further embodiment, the bridge molecule system is selected from MFG-E8, Gas6, thrombospondin (TSP) -1 and TSP-2. In the embodiments of the present invention, the cells are precursor cells. In a specific embodiment of the present invention, the cells are postpartum-derived cells. In an embodiment of the invention, the postpartum-derived cell lines are isolated from human umbilical cord tissue or placental tissue that is substantially free of blood.
在如上文所述之本發明之實施例中,產後衍生細胞群具有以下特徵:在經塗布或未經塗布的組織培養容器上附著並擴 增,其中經塗布之組織培養容器包含明膠、層黏蛋白、膠原蛋白、聚鳥胺酸、玻璃連接蛋白、或纖連蛋白素之塗層;生產波形蛋白與α-平滑肌肌動蛋白;且係對HLA-A、HLA-B、HLA-C陽性,及對HLA-DR、HLA-DP、HLA-DQ陰性。 In the embodiment of the present invention as described above, the postpartum-derived cell population has the following characteristics: it is attached and expanded on the coated or uncoated tissue culture container, wherein the coated tissue culture container contains gelatin, lamellar adhesive Coating of protein, collagen, polyguanylic acid, vitronectin, or fibronectin; production of vimentin and α-smooth muscle actin; and positive for HLA-A, HLA-B, HLA-C, And negative for HLA-DR, HLA-DP, HLA-DQ.
在如上文所述之本發明之實施例中,視網膜變性、視網膜病變或視網膜/黃斑病症為年齡相關性黃斑變性。在一替代實施例中,視網膜變性、視網膜病變、或視網膜/黃斑病症為乾性年齡相關性黃斑變性。 In the embodiments of the present invention as described above, the retinal degeneration, retinopathy, or retinal / macular disorder is age-related macular degeneration. In an alternative embodiment, the retinal degeneration, retinopathy, or retinal / macular disorder is dry age-related macular degeneration.
在如上文所述之本發明之實施例中,光受體細胞損失係藉由抑制光受體細胞之細胞凋亡來減少或預防。在實施例中,光受體細胞損失係藉由刺激脫落光受體片段之吞噬來減少或預防。 In the embodiments of the present invention as described above, the loss of photoreceptor cells is reduced or prevented by inhibiting apoptosis of photoreceptor cells. In an embodiment, photoreceptor cell loss is reduced or prevented by stimulating phagocytosis of shed photoreceptor fragments.
圖1A至圖1B顯示預培養於具有血清之hUTC CM1製劑對失養性RPE吞噬之作用。圖1A.色素失養性RPE係經CM1製劑(具有血清)預培養。至於對照組,將褐色頭套狀正常與色素失養性RPE預培養於對照培養基(DMEM:F12培養基+10% FBS+Pen(50U/ml)/Strep(50μg/ml))。培養時間為16小時。(tan N:褐色頭套狀正常RPE;pig D:色素失養性RPE;con:對照組;M:培養基;CM:條件培養基;w:具有)。值為一個樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本11或12個;n:所計數的視野數目);與pig D對照組比較,p<0.05。 圖1B.失養性RPE係經CM1(具有血清)預培養。至於對照組,將正常與失養性RPE預培養於具有血清之對照培養基中。培養時間為24小時。(N:正常RPE;D:失養性RPE;D(1):三重複1;D(2):三重複2;D(3):三重複3;con:對照組;M:培養基;CM:條件培養基;w:具有)。值為一個樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本11、12或13個;n:所計數的視野數目)。與D對照組比較,p<0.05。 Figures 1A-1B show the effect of pre-cultivation of hUTC CM1 preparation with serum on phagocytosis of deferent RPE. Figure 1A. Pigment-dystrophic RPE is pre-cultured with CM1 preparation (with serum). As for the control group, the brown hooded normal and pigment-dystrophic RPE were pre-cultured in a control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)). The cultivation time is 16 hours. (tan N: brown cape-shaped normal RPE; pig D: pigment-dystrophic RPE; con: control group; M: medium; CM: conditioned medium; w: with). The value is the average value of the number of ROS phagocytized by a sample in the counting field ± SD (n = 11 or 12 per sample; n: number of fields counted); compared with the pig D control group, p <0.05. Figure 1B. The dystrophic RPE line is pre-cultured with CM1 (with serum). As for the control group, normal and deferent RPE were pre-cultured in control medium with serum. The cultivation time is 24 hours. (N: normal RPE; D: dystrophy RPE; D (1): triple repeat 1; D (2): triple repeat 2; D (3): triple repeat 3; con: control group; M: medium; CM: Conditioned medium; w: with). The value is the average value of the number of ROS phagocytosed by a sample in the counting field ± SD (n = 11, 12, or 13 per sample; n: number of fields counted). Compared with the D control group, p <0.05.
圖2顯示預培養於不含血清之hUTC CM1製劑對失養性RPE吞噬之作用。色素失養性RPE係經CM1(不含血清)預培養。至於對照組,將褐色頭套狀正常與色素失養性RPE預培養於不含血清之對照培養基中。培養時間為24小時。(tan N:褐色頭套狀正常RPE;pig D:色素失養性RPE;con:對照組。M:培養基;CM:條件培養基;wo:不含。值為一個樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本9、10、11或12個;n:所計數的視野數目);與pig D對照組比較,p<0.05。 Figure 2 shows the effect of pre-cultured serum-free hUTC CM1 preparation on phagocytosis of deferent RPE. The pigment-dystrophic RPE is pre-cultured with CM1 (without serum). As for the control group, the brown hooded normal and pigment-dystrophic RPE were pre-cultured in a control medium without serum. The cultivation time is 24 hours. (tan N: brown hood-like normal RPE; pig D: pigment-dystrophic RPE; con: control group. M: medium; CM: conditioned medium; wo: no. The value is the number of ROS phagocytosed by a sample in the counting field The mean ± SD (n = 9, 10, 11 or 12 per sample; n: number of fields counted); compared with the pig D control group, p <0.05.
圖3A至圖3D顯示預培養於hUTC CM對吞噬之作用。(圖3A)預培養於hUTC CM2對吞噬的作用。色素失養性RPE係經CM2預培養。至於對照組,將褐色頭套狀正常與色素失養性RPE預培養於對照培養基中。培養時間為24小時。(tan N:褐色頭套狀正常RPE;pig D:色素失養性RPE;con:對照組。M:培養基;CM:條件培養基)。值為一個樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本8或10個;n:所計數的視 野數目)。(圖3B至圖3D)預培養於hUTC CM3對吞噬之作用。失養性RPE係經CM3預培養。至於對照組,將正常與失養性RPE預培養於對照培養基中。培養時間為24小時。圖3B.(N:正常RPE;D:失養性RPE;con:對照組;M:培養基;CM:條件培養基)。值為三重複樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本5至12個;n:所計數的視野數目);與D+con M對照組比較,p<0.05。圖3C.正常RPE對照組係來自CM3測試1。(N:正常RPE;D:失養性RPE;con:對照組;M:培養基;CM:條件培養基)。值為一個樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本11或14個;n:所計數的視野數目);與D+con M對照組比較,p<0.05。圖3D.(N:正常RPE;N1:來自培養物之正常RPE;N2:來自N2培養物之正常RPE;tan N:褐色頭套狀正常RPE;D:失養性RPE;con:對照組;M:培養基;CM:條件培養基)。值為一個樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本12或13個;n:所計數的視野數目);與D+con M對照組比較,p<0.05。 Figures 3A to 3D show the effect of CM pre-cultured in hUTC on phagocytosis. (Figure 3A) The effect of CM2 pre-cultured in hUTC on phagocytosis. The pigment-dystrophic RPE line is pre-cultured with CM2. As for the control group, the brown hooded normal and pigment-dystrophic RPE were pre-cultured in the control medium. The cultivation time is 24 hours. (tan N: brown hooded normal RPE; pig D: pigment-dystrophic RPE; con: control group. M: medium; CM: conditioned medium). The value is the average of the number of ROS phagocytosed by a sample in the counting field ± SD (n = 8 or 10 per sample; n: number of fields counted). (Figure 3B to Figure 3D) The effect of CM3 pre-cultured on phagocytosis. Infertile RPE is pre-cultured with CM3. As for the control group, normal and deferent RPE were pre-cultured in the control medium. The cultivation time is 24 hours. Figure 3B. (N: normal RPE; D: defertile RPE; con: control group; M: medium; CM: conditioned medium). Values are the average of the number of ROS phagocytosed by the replicate samples in the counting field ± SD (n = 5 to 12 per sample; n: number of fields counted); compared with the D + con M control group, p <0.05 . Figure 3C. Normal RPE control line from CM3 test 1. (N: normal RPE; D: dystrophic RPE; con: control group; M: medium; CM: conditioned medium). The value is the average value of the number of ROS phagocytosed by a sample in the counting field ± SD (n = 11 or 14 per sample; n: number of fields counted); compared with the D + con M control group, p <0.05. Figure 3D. (N: normal RPE; N1: normal RPE from culture; N2: normal RPE from N2 culture; tan N: brown hooded normal RPE; D: defertile RPE; con: control group; M: Medium; CM: conditioned medium). The value is the average value of the number of ROS phagocytized by a sample in the counting field ± SD (n = 12 or 13 per sample; n: number of fields counted); compared with the D + con M control group, p <0.05.
圖4A至圖4C顯示hUTC對體外RCS RPE細胞中之吞噬的作用。RCS RPE細胞與接種於跨孔(transwell)中的hUTC共培養(圖4A)、或以hUTC CM培養(圖4B)24小時且隨後進行吞噬檢定。N:正常RPE;D:失養性RCS RPE;CM,條件培養基。在與hUTC共培養或經hUTC CM培養之RCS RPE中觀察到吞 噬增加。(圖4C)光受體OS係以hUTC CM培養24小時且隨後饋至RCS RPE細胞以在hUTC CM不存在下進行吞噬檢定。經hUTC CM處理之OS使RCS RPE之吞噬恢復。數據代表平均值±SEM(n=3)。****p<0.0001。 4A to 4C show the effect of hUTC on phagocytosis in RCS RPE cells in vitro. RCS RPE cells were co-cultured with hUTC seeded in transwell (Figure 4A) or with hUTC CM (Figure 4B) for 24 hours and then subjected to phagocytosis assay. N: normal RPE; D: defertile RCS RPE; CM, conditioned medium. Increased phagocytosis was observed in RCS RPE co-cultured with hUTC or cultured with hUTC CM. (Figure 4C) The photoreceptor OS line was cultured with hUTC CM for 24 hours and then fed to RCS RPE cells for phagocytosis assay in the absence of hUTC CM. The OS treated with hUTC CM restored RCS RPE phagocytosis. The data represent the mean ± SEM (n = 3). **** p <0.0001.
圖5A至圖5B顯示RTK配體BDNF或HB-EGF之檢定結果。失養性RPE細胞係以BDNF(200ng/ml)(圖5A)或HB-EGF(200ng/ml)(圖5B)培養於MEM+5% FBS(MEM5)中24h。至於陽性對照組,將失養性RPE細胞培養於CM3中24h。至於其他對照組,將正常及失養性RPE培養於MEM5中24h並進行吞噬檢定。(N:正常RPE;D:失養性RPE;con:對照組;M:培養基;CM:條件培養基)。值為二重複或三重複樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本10、11或12個;n:所計數的視野數目);與D對照組比較,p<0.05。 5A to 5B show the results of the RTK ligand BDNF or HB-EGF test. The dystrophic RPE cell line was cultured in MEM + 5% FBS (MEM5) for 24h with BDNF (200ng / ml) ( Figure 5A ) or HB-EGF (200ng / ml) ( Figure 5B ). As for the positive control group, the dystrophic RPE cells were cultured in CM3 for 24h. As for the other control groups, normal and deferent RPE were cultured in MEM5 for 24 hours and subjected to phagocytosis test. (N: normal RPE; D: dystrophic RPE; con: control group; M: medium; CM: conditioned medium). The value is the average of the number of ROS phagocytosed in the field of counts of duplicate or triple repeat samples ± SD (n = 10, 11 or 12 per sample; n: number of counted fields); compared with the D control group, p <0.05.
圖6A至圖6E顯示RTK配體PDGF-DD、蝶素(ephrin)A4、及HGF之檢定結果。失養性RPE細胞係以PDGF-DD(圖6A、圖6B)、蝶素A4(圖6C)或HGF(200ng/ml)(圖6D、圖6E)培養於MEM5中24h且隨後添加ROS於含有PDGF-DD、蝶素A4或HGF之MEM5中以進行吞噬檢定(當添加ROS時培養基未更換)。至於陽性對照組,將失養性RPE細胞培養於CM3中24h。至於其他對照組,將正常及失養性RPE培養於MEM5中24h並進行吞噬檢定。(N:正常RPE;D:失養性RPE;con:對照組;M:培養基;CM:條件培養基)。值為二重複或三重複 樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本10、11或12個;n:所計數的視野數目);與D對照組比較,p<0.05。 6A to 6E show the test results of RTK ligand PDGF-DD, ephrin A4, and HGF. The dystrophic RPE cell line was cultured in MEM5 with PDGF-DD ( Figure 6A, Figure 6B ), Pterin A4 ( Figure 6C ) or HGF (200ng / ml) ( Figure 6D, Figure 6E ) for 24h and then added ROS to the PDGF containing -DD5, Pterin A4 or HGF in MEM5 for phagocytosis assay (the medium was not changed when ROS was added). As for the positive control group, the dystrophic RPE cells were cultured in CM3 for 24h. As for the other control groups, normal and deferent RPE were cultured in MEM5 for 24 hours and subjected to phagocytosis test. (N: normal RPE; D: dystrophic RPE; con: control group; M: medium; CM: conditioned medium). The value is the average of the number of ROS engulfed in the counting field of the duplicate or triple samples ± SD (n = 10, 11 or 12 per sample; n: the number of counted fields); compared with the D control group, p <0.05.
圖7A至圖7B顯示RTK配體蝶素B2之檢定結果。失養性RPE細胞係以蝶素B2(200ng/ml)培養。至於陽性對照組,將失養性RPE細胞培養於CM3中。至於其他對照組,將正常與失養性RPE培養於MEM5中24h且進行吞噬檢定。(N:正常RPE;D:失養性RPE;con:對照組;M:培養基;CM:條件培養基)。值為二重複或三重複樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本10、11或12個;n:所計數的視野數目);與D對照組比較,p<0.05。 7A to 7B show the results of RTK ligand Pterin B2 assay. The dystrophic RPE cell line was cultured with Pterin B2 (200ng / ml). As for the positive control group, the dystrophic RPE cells were cultured in CM3. As for the other control groups, normal and deferent RPE were cultured in MEM5 for 24h and subjected to phagocytosis test. (N: normal RPE; D: dystrophic RPE; con: control group; M: medium; CM: conditioned medium). The value is the average of the number of ROS engulfed in the counting field of the duplicate or triple samples ± SD (n = 10, 11 or 12 per sample; n: the number of counted fields); compared with the D control group, p <0.05.
圖8A至圖8C顯示經內皮素-1、TGF-β1、或IL-6處理之失養性RPE細胞。(圖8A)對內皮素-1或TGF-β1(以200ng/mL)相較於正常對照組之吞噬進行檢定。(圖8B、圖8C)失養性RPE細胞係以200ng/mL之重組人類內皮素-1、TGF-β1、或IL-6培養且檢定其吞噬。hUTC CM3係用作陽性對照組。至於其他對照,將正常與失養性RPE培養於MEM5中24h且進行吞噬檢定。(N:正常RPE;D:失養性RPE)。值為每個樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本10個;n:所計數的視野數目)。 8A to 8C show dystrophic RPE cells treated with endothelin-1, TGF-β1, or IL-6. (Figure 8A) Verification of phagocytosis of endothelin-1 or TGF-β1 (at 200 ng / mL) compared to the normal control group. (Figure 8B, Figure 8C) The dystrophic RPE cell line was cultured with 200ng / mL recombinant human endothelin-1, TGF-β1, or IL-6 and tested for phagocytosis. The hUTC CM3 line was used as a positive control group. As for other controls, normal and deferent RPE were cultured in MEM5 for 24h and subjected to phagocytosis assay. (N: normal RPE; D: infertile RPE). The value is the average value of the number of ROS phagocytosed by each sample in the counting field ± SD (n = 10 per sample; n: number of fields counted).
圖9顯示將失養性RPE細胞饋以用各種濃度之玻璃連接蛋白預培養的ROS,並檢定其以及正常對照組之吞噬。ROS係以對照 培養基(DMEM+10% FBS)或CM3預培養。同時,ROS係以各種濃度之人類重組玻璃連接蛋白(4、2、1、0.5ug/ml)分別預培養於MEM20中。至於對照組,將單獨正常RPE或單獨失養性RPE培養於MEM20中,隨後在未處理ROS(再懸浮於MEM20中且饋至RPE細胞)存在下更換成MEM5以進行吞噬檢定。(N:正常RPE;D:失養性RPE;Con M:對照培養基;V:玻璃連接蛋白)。值為每個樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本10個;n:所計數的視野數目)。 Fig. 9 shows that dystrophic RPE cells were fed with ROS pre-cultured with various concentrations of vitronectin, and the phagocytosis of the normal control group was examined. ROS lines were pre-cultured with control medium (DMEM + 10% FBS) or CM3. At the same time, ROS was pre-cultured in MEM20 with various concentrations of human recombinant glass connexin (4, 2, 1, 0.5ug / ml). As for the control group, normal RPE alone or deprived RPE alone was cultured in MEM20, and then replaced with MEM5 in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) for phagocytosis assay. (N: normal RPE; D: dystrophic RPE; Con M: control medium; V: glass connexin). The value is the average value of the number of ROS phagocytosed by each sample in the counting field ± SD (n = 10 per sample; n: number of fields counted).
圖10顯示在hUTC中所識別之RTK配體之基因表現水準。總mRNA係萃取自hUTC且執行RNA-Seq以識別及定量hUTC中之RTK配體基因表現。經識別之RTK配體係基於對應RTK次家族分類且根據其FPKM值分級。偵測15個RTK次家族之多個RTK配體於hUTC中之基因表現。 Figure 10 shows the gene expression level of RTK ligands identified in hUTC. Total mRNA was extracted from hUTC and RNA-Seq was performed to identify and quantify RTK ligand gene expression in hUTC. The identified RTK matching system is classified based on the corresponding RTK subfamily and graded according to its FPKM value. Detect the gene expression of multiple RTK ligands in 15 RTK subfamilies in hUTC.
圖11A至圖11G顯示在hUTC CM中所量測之選定RTK配體水準。(圖11A至圖11F)RTK配體之水準係與NHDF及ARPE-19條件培養基中之配體水準比較。相較於NHDF及ARPE-19條件培養基,BDNF係以高水準分泌於hUTC條件培養基中(圖11A)。相較於NHDF CM,NT3在hUTC CM中之水準係高,然而NT3於ARPE-19條件培養基及對照培養基中之量不可偵測(圖11B)。相較於NHDF及ARPE-19條件培養基,HGF係以高水準分泌於hUTC CM中(圖11C)。相較於NHDF及ARPE-19條件培養基,PDGF-CC及PDGF-DD在hUTC條件培養基中之 水準係低(分別為圖11D與圖11E)。GDNF係分泌於hUTC及NHDF條件培養基中,但在ARPE-19條件培養基中僅痕量(圖11F)。所有值為三重複樣本之平均值±SD,除了NT3為二重複樣本之平均值±SD。(圖11G)藉由ELISA所量測之RTK配體水準。(所顯示之數據為平均值±SEM;n=3)。 Figures 11A to 11G show the selected RTK ligand levels measured in hUTC CM. (FIG. 11A to FIG. 11F) RTK ligand levels are compared with those in NHDF and ARPE-19 conditioned media. Compared with NHDF and ARPE-19 conditioned medium, the BDNF line was secreted at high levels in hUTC conditioned medium ( Figure 11A ). Compared with NHDF CM, the level of NT3 in hUTC CM is high, however, the amount of NT3 in ARPE-19 conditioned medium and control medium is undetectable ( Figure 11B ). Compared with NHDF and ARPE-19 conditioned medium, HGF lines are secreted at high levels in hUTC CM ( Figure 11C ). Compared with NHDF and ARPE-19 conditioned medium, the levels of PDGF-CC and PDGF-DD in hUTC conditioned medium are low ( Figure 11D and Figure 11E, respectively ). The GDNF line is secreted in hUTC and NHDF conditioned medium, but only in trace amounts in ARPE-19 conditioned medium ( Figure 11F ). All values are the mean ± SD of triplicate samples, except NT3 is the mean ± SD of duplicate samples. (FIG. 11G) RTK ligand level measured by ELISA. (The data shown is the mean ± SEM; n = 3).
圖12A至圖12E顯示在hUTC CM中所量測之橋分子水準。(圖12A至圖12E)。橋分子之水準係與NHDF及ARPE-19條件培養基比較。圖12A顯示hUTC、ARPE-19及NHDF條件培養基中之MFG-E8水準。值為二重複或三重複樣本之平均值±SD。圖12B顯示hUTC、ARPE-19及NHDF條件培養基中之Gas6水準。值為二重複樣本之平均值±SD。圖12C顯示hUTC、ARPE-19及NHDF條件培養基中之TSP-1水準。值為二重複樣本之平均值±SD。圖12D顯示hUTC、ARPE-19及NHDF條件培養基中之TSP-2水準。值為二重複樣本之平均值±SD。(圖12E)藉由ELISA所量測之橋分子水準。所顯示之數據為平均值±SEM(對於TSP-1及TSP-2,n=2;對於所有MFG-E8,n=3)。 Figures 12A to 12E show the level of bridge molecules measured in hUTC CM. (Figures 12A to 12E). The level of bridge molecules is compared with NHDF and ARPE-19 conditioned medium. Figure 12A shows MFG-E8 levels in hUTC, ARPE-19 and NHDF conditioned medium. The value is the mean ± SD of duplicate or triple samples. Figure 12B shows Gas6 levels in hUTC, ARPE-19 and NHDF conditioned medium. Values are the mean ± SD of duplicate samples. Figure 12C shows the levels of TSP-1 in conditioned medium of hUTC, ARPE-19 and NHDF. Values are the mean ± SD of duplicate samples. Figure 12D shows TSP-2 levels in hUTC, ARPE-19 and NHDF conditioned medium. Values are the mean ± SD of duplicate samples. (Fig. 12E) The level of bridge molecules measured by ELISA. The data shown is the mean ± SEM (n = 2 for TSP-1 and TSP-2; n = 3 for all MFG-E8).
圖13A至圖13D展示用hUTC條件培養基(CM)預培養失養性RPE細胞對吞噬之作用。對於對照組,將單獨正常RPE或單獨失養性RPE預培養於其常規生長培養基MEM20(MEM+20% FBS)中。同時,失養性RPE亦經CM3預培養。此外,測試經hUTC CM3預培養之ROS。值為每個樣本在計數視野中經吞噬之ROS數目之平均值±SD。圖13A,n=每樣本10至20個;圖 13B為原始數據。圖13C,n=每樣本12個且各樣本係二重複。(n:所計數的視野數目);圖13D為原始數據。 Figures 13A to 13D show the effect of precultivation of dystrophic RPE cells with hUTC conditioned medium (CM) on phagocytosis. For the control group, normal RPE alone or deprived RPE alone were pre-cultured in their conventional growth medium MEM20 (MEM + 20% FBS). At the same time, dystrophic RPE was also pre-cultured with CM3. In addition, ROS pre-cultured with hUTC CM3 was tested. The value is the mean ± SD of the number of phagocytized ROS of each sample in the counting field. Figure 13A , n = 10 to 20 per sample; Figure 13B is the original data. Figure 13C , n = 12 per sample and each sample is duplicated. (n: number of counted fields of view); Figure 13D is the original data.
圖14A至圖14K顯示橋分子及RTK配體對RCS RPE細胞吞噬視桿細胞外節(ROS)之作用。ROS係以對照培養基(DMEM+10% FBS)或hUTC條件培養基預培養。同時,ROS係以各種濃度之人類重組MFG-E8(圖14A)、Gas6(圖14B)、TSP-1(圖14C)、或TSP-2(圖14D)預培養於對照培養基中。至於對照組,將單獨正常RPE或單獨失養性RPE培養以用於吞噬檢定。(N:正常RPE;N+ROS:在吞噬檢定期間正常RPE細胞饋以未處理之ROS;D:失養性RPE細胞;D+ROS:在吞噬檢定期間失養性RPE細胞饋以未處理之ROS;Con M:對照培養基;D+ROS+Con:失養性RPE細胞饋以經對照培養基預培養之ROS;CM4:第4批hUTC條件培養基)。D+ROS+CM4:失養性RPE細胞饋以經CM4預培養之ROS;D+ROS+MFG-E8:失養性RPE細胞饋以經MFG-E8預培養之ROS。值為每個樣本在計數視野中經吞噬之ROS數目之平均值±SD(n=每樣本10個;n:所計數的視野數目)。*P<0.001,經MFG-E8、Gas6、TSP-1或TSP-2預處理之D+ROS與D+ROS+ConM、及D+ROS比較;**P<0.0001,D+ROS+CM4與D+ROS、及D+ROS+ConM比較。 (圖14E至圖14H)光受體OS係以重組人類MFG-E8(圖14E)、Gas6(圖14F)、TSP-1(圖14G)、或TSP-2(圖14H)培養24小時且隨後饋至RCS RPE細胞以在hUTC CM不存在下進行吞 噬檢定。經hUTC CM預培養之OS係用作檢定之陽性對照。RCS RPE細胞對OS之吞噬係以劑量依賴性方式受到MFG-E8、Gas6、TSP-1或TSP-2之救援。(圖14I至圖14K)RCS RPE細胞係經重組人類BDNF(圖14I)、HGF(圖14J)或GDNF(圖14K)培養24小時,且隨後進行吞噬檢定。經hUTC CM培養之RCS RPE係用作檢定之陽性對照。BDNF、HGF或GDNF劑量依賴性地增加RCS RPE細胞中之吞噬。數據代表平均值±SEM(n=3)。****p<0.0001,***p<0.001,**p<0.01,*p<0.05,n.s.不顯著。 Figures 14A to 14K show the effect of bridge molecules and RTK ligands on RCS RPE cells phagocytosis of the rod outer segment (ROS). ROS lines were pre-cultured with control medium (DMEM + 10% FBS) or hUTC conditioned medium. At the same time, ROS lines were pre-cultured in control medium with various concentrations of human recombinant MFG-E8 ( Figure 14A ), Gas6 ( Figure 14B ), TSP-1 ( Figure 14C ), or TSP-2 ( Figure 14D ). As for the control group, normal RPE alone or deprived RPE alone were cultured for phagocytosis assay. (N: normal RPE; N + ROS: normal RPE cells are fed with untreated ROS during the phagocytosis test; D: dystrophic RPE cells; D + ROS: dystrophic RPE cells are fed with untreated ROS during the phagocytosis test; Con M: control medium; D + ROS + Con: defertile RPE cells were fed with ROS pre-cultured in control medium; CM4: batch 4 hUTC conditioned medium). D + ROS + CM4: dystrophic RPE cells fed ROS pre-cultured with CM4; D + ROS + MFG-E8: dystrophic RPE cells fed ROS pre-cultured with MFG-E8. The value is the average value of the number of ROS phagocytosed by each sample in the counting field ± SD (n = 10 per sample; n: number of fields counted). * P <0.001, compared with D + ROS and D + ROS + ConM, and D + ROS pretreated with MFG-E8, Gas6, TSP-1 or TSP-2; ** P <0.0001, D + ROS + CM4 and Comparison of D + ROS and D + ROS + ConM. (FIG. 14E to FIG. 14H) The photoreceptor OS line was cultured with recombinant human MFG-E8 (FIG. 14E) , Gas6 (FIG. 14F) , TSP-1 (FIG. 14G) , or TSP-2 (FIG. 14H) for 24 hours and then Feed to RCS RPE cells for phagocytosis assay in the absence of hUTC CM. The OS pre-cultured with hUTC CM is used as a positive control for the test. The phagocytosis of OS by RCS RPE cells was rescued by MFG-E8, Gas6, TSP-1 or TSP-2 in a dose-dependent manner. ( FIG. 14I to FIG. 14K) RCS RPE cell lines were cultured with recombinant human BDNF ( FIG. 14I ), HGF ( FIG. 14J ), or GDNF ( FIG. 14K ) for 24 hours, and then subjected to phagocytosis assay. RCS RPE cultured with hUTC CM is used as a positive control for the test. BDNF, HGF or GDNF dose-dependently increased phagocytosis in RCS RPE cells. The data represent the mean ± SEM (n = 3). **** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, ns is not significant.
圖15A至圖15C.用於RCS RPE中hUTC誘導之吞噬救援的RTK配體及橋分子。(圖15A)自未轉染之hUTC及經siRNA轉染之hUTC收集之細胞培養物上清液的ELISA。(圖15B)RTK配體BDNF、HGF及GDNF之表現受到hUTC之siRNA轉染而靜默。收集基因剔除(Knockdown,KD)hUTC CM。RCS RPE係經KD hUTC CM培養24小時且隨後進行吞噬檢定。當BDNF、HGF或GDNF經剔除時,hUTC對RCS RPE吞噬之作用係經廢除。(圖15C)hUTC中橋分子MFG-E8、TSP-1及TSP-2之表現受到siRNA轉染而靜默。收集KD hUTC CM。將RCS RPE饋以經KD hUTC CM預培養24小時之OS並進行吞噬檢定。MFG-E8、TSP-1、或TSP-2之剔除減少RCS RPE中hUTC媒介之OS吞噬救援。製備自未轉染及經淩亂(scrambled)siRNA轉染之hUTC的CM係用作為對照組。數據代表平均值±SEM,(B)及(C)之n=3,未 轉染、模擬及淩亂siRNA轉染hUTC CM ELISA(A)之n=6。****p<0.0001,***p<0.001,**p<0.01,*p<0.05,n.s.不顯著。 Figures 15A to 15C. RTK ligands and bridge molecules used for hUTC-induced phagocytosis rescue in RCS RPE. (Figure 15A) ELISA of cell culture supernatant collected from untransfected hUTC and siRNA transfected hUTC. (Figure 15B) The performance of RTK ligands BDNF, HGF and GDNF was silenced by hUTC siRNA transfection. Collect Knockdown (Knockdown, KD) hUTC CM. The RCS RPE line was cultured with KD hUTC CM for 24 hours and then subjected to phagocytosis assay. When BDNF, HGF or GDNF is eliminated, the effect of hUTC on RCS RPE phagocytosis is abolished. (Figure 15C) The performance of the bridge molecules MFG-E8, TSP-1 and TSP-2 in hUTC was silenced by siRNA transfection. Collect KD hUTC CM. RCS RPE was fed with OS pre-cultured with KD hUTC CM for 24 hours and subjected to phagocytosis test. The elimination of MFG-E8, TSP-1, or TSP-2 reduces OS phagocytosis rescue of hUTC media in RCS RPE. CM lines prepared from untransfected and scrambled siRNA transfected hUTC were used as a control group. The data represent the mean ± SEM, n = 3 for (B) and (C), and n = 6 for hUTC CM ELISA (A) transfected with untransfected, simulated, and messy siRNA. **** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, ns is not significant.
圖16A至圖16D. hUTC分泌之橋分子結合至光受體OS。OS的免疫螢光(IF)染色,OS係經個別重組人類MFG-E8(124ng/mL)、Gas6(8.75ng/mL)、TSP-1(1.2ng/mL)或TSP-2(238ng/mL)(圖16A)、或hUTC CM(圖16B)、或對照培養基(圖16C)培養24小時,稍後進行視紫質與橋分子的雙重IF染色,視紫質係經Alexa Fluor 568共軛抗視紫質抗體染色,橋分子係經Alexa Fluor 488共軛抗MFG-E8、抗Gas6、抗TSP-1、抗TSP-2、小鼠IgG2A或小鼠IgG2B同型對照抗體染色。視紫質經染色之OS粒子亦呈重組橋分子蛋白質之各者或存在於hUTC CM中之分泌橋分子的陽性染色。(圖16D)抗視紫質抗體之特異性藉由以Alexa Fluor 568共軛抗視紫質抗體及Alexa Fluor 488共軛小鼠IgG2b,x同型對照抗體雙重IF染色OS來確認。上圖(圖16A至圖16D),橋分子與同型抗體染色;下圖(圖16A至圖16D),視紫質抗體染色。 Figures 16A-16D. The bridge molecule secreted by hUTC binds to the photoreceptor OS. Immunofluorescence (IF) staining of OS, OS was individually recombinant human MFG-E8 (124ng / mL), Gas6 (8.75ng / mL), TSP-1 (1.2ng / mL) or TSP-2 (238ng / mL) ) ( Figure 16A ), or hUTC CM ( Figure 16B ), or control medium ( Figure 16C ) for 24 hours, and then double IF staining of rhodopsin and bridge molecules, rhodopsin is conjugated by Alexa Fluor 568 The rhodopsin antibody was stained and the bridge molecule was stained with Alexa Fluor 488 conjugated anti-MFG-E8, anti-Gas6, anti-TSP-1, anti-TSP-2, mouse IgG2A or mouse IgG2B isotype control antibody. The rhodopsin-stained OS particles also showed positive staining for each of the recombinant bridge molecule proteins or secretory bridge molecules present in hUTC CM. ( Figure 16D ) The specificity of anti-rhodopsin antibodies was confirmed by double IF staining of OS with Alexa Fluor 568 conjugated anti-rhodopsin antibody and Alexa Fluor 488 conjugated mouse IgG2b, x isotype control antibody. Upper panel ( Figures 16A-16D ), bridge molecules and isotype antibody staining; lower panel (Figures 16A-16D), rhodopsin antibody staining.
圖17A至圖17F顯示hUTC及hUTC條件培養基防止氧化應力或損傷。圖17A至圖17B說明hUTC條件培養基防止含A2E之RPE細胞在430nm照射後無存活。(圖17A)。細胞死亡是使用雙色螢光檢定來檢定。hUTC條件培養基及非條件對照培養基(250μL/孔)係與富含A2E之ARPE-19細胞一起培養7天。非存活細胞之百分比係藉由雙色螢光檢定測定;5重複。圖17B 顯示來自5%及10% FBS處理之彙集數據。值為平均值+/- SEM。p<0.05;單因子ANOVA及紐柯(Newman Keuls)多重比較測試。圖17C至17D說明hUTC條件培養基防止ARPE-19細胞與A2E光氧化相關聯之細胞存活性減少。(圖17C)。藉由MTT檢定細胞存活性。hUTC條件培養基及非條件對照培養基(250μL/孔)係與富含A2E之ARPE-19細胞一起培養(7天,37℃,5% CO2,5% FBS)。條柱高度指出MTT吸光度且反映細胞存活性。圖17D顯示來自5%及10% FBS處理之彙集數據。值為平均值+/- SEM;4個重複/2個實驗。*p>0.05;**p<0.05;單因子ANOVA及紐柯(Newman Keuls)多重比較測試。圖17E至17F說明hUTC條件培養基防止ARPE-19細胞與急性H2O2相關聯之細胞存活性減少。藉由MTT(圖17E)及結晶紫(圖17F)檢定細胞存活性。y軸代表在550nm下之校正OD讀數。數據係表示為平均值±標準偏差。p<0.05,利用二因子ANOVA。 17A to 17F show that hUTC and hUTC conditioned medium prevent oxidative stress or damage. 17A to 17B illustrate that hUTC conditioned medium prevents APE-containing RPE cells from surviving after 430 nm irradiation. (Figure 17A). Cell death is detected using a two-color fluorescent test. The hUTC conditioned medium and unconditioned control medium (250 μL / well) were cultured with A2E-rich ARPE-19 cells for 7 days. The percentage of non-viable cells was determined by two-color fluorescence assay; 5 repeats. Figure 17B shows pooled data from 5% and 10% FBS processing. Values are average +/- SEM. p <0.05; single factor ANOVA and Newman Keuls multiple comparison test. Figures 17C to 17D illustrate that hUTC conditioned medium prevents ARPE-19 cells from reducing cell viability associated with A2E photooxidation. (Figure 17C). Cell viability was tested by MTT. hUTC conditioned medium and unconditioned control medium (250 μL / well) were cultured with ARPE-19 cells rich in A2E (7 days, 37 ° C, 5% CO 2 , 5% FBS). Bar height indicates MTT absorbance and reflects cell viability. Figure 17D shows pooled data from 5% and 10% FBS processing. Values are mean +/- SEM; 4 replicates / 2 experiments. * p >0.05; ** p <0.05; single factor ANOVA and Newman Keuls multiple comparison test. 17E to 17F illustrate that hUTC conditioned medium prevents ARPE-19 cells from reducing cell viability associated with acute H 2 O 2 . Cell viability was verified by MTT (Figure 17E) and crystal violet (Figure 17F) . The y-axis represents the corrected OD reading at 550nm. The data system is expressed as mean ± standard deviation. p <0.05, using two-factor ANOVA.
圖18顯示人類RPE及大鼠RPE吞噬之免疫螢光顯微照片。 Figure 18 shows immunofluorescence micrographs of human RPE and rat RPE phagocytosis.
圖19顯示人類RPE及大鼠RPE吞噬之時程免疫螢光顯微照片。 Figure 19 shows immunofluorescence micrographs of the time course of human RPE and rat RPE phagocytosis.
圖20顯示以大鼠RPE吞噬人類ROS之免疫螢光顯微照片。 Figure 20 shows immunofluorescence micrographs of human ROS engulfed by rat RPE.
圖21顯示人類RPE(樣本15-02-032)之細胞角蛋白免疫染色。 Figure 21 shows cytokeratin immunostaining of human RPE (sample 15-02-032).
圖22顯示San Diego 1「乾性」AMD RPE之細胞角蛋白免疫染色。 Figure 22 shows the cytokeratin immunostaining of San Diego 1 "dry" AMD RPE.
圖23顯示人類濕性AMD RPE(樣本15-10-021)之細胞角蛋白免疫染色。 Figure 23 shows cytokeratin immunostaining of human wet AMD RPE (sample 15-10-021).
圖24顯示ND08333「AMD」RPE之細胞角蛋白免疫染色。 Figure 24 shows cytokeratin immunostaining of ND08333 "AMD" RPE.
圖25A及圖25B顯示來自不同年齡個體的RPE吞噬水準比較。 Figures 25A and 25B show the comparison of RPE phagocytic levels from individuals of different ages.
圖26顯示正常RPE與AMD(SD1 L & R(「乾性」)、15-10-021(「濕性」))RPE對15-10-021上帶ROS之吞噬比較。 Figure 26 shows the comparison of phagocytosis of ROS on 15-10-021 by normal RPE and AMD (SD1 L & R ("dry"), 15-10-021 ("wet")) RPE.
圖27顯示人類RPE(樣本15-08-074)的人類ROS劑量反應測試結果。 Figure 27 shows the human ROS dose response test results for human RPE (sample 15-08-074).
圖28顯示人類RPE(樣本15-11-098)的人類ROS劑量反應測試結果。 Figure 28 shows the human ROS dose response test results for human RPE (Samples 15-11-098).
圖29顯示在經或未經CM下AMD RPE及正常RPE對人類眼睛15-09-027上帶ROS(包括人類眼睛15-04-001 RPE)之吞噬。 Figure 29 shows the phagocytosis of ROS on human eye 15-09-027 (including human eye 15-04-001 RPE) by AMD RPE and normal RPE with or without CM.
圖30顯示RTK配體GDNF對於AMD RPE吞噬之作用。 Figure 30 shows the effect of RTK ligand GDNF on AMD RPE phagocytosis.
圖31顯示RTK配體HGF對於「濕性」AMD RPE(15-10-021)吞噬之作用。 Figure 31 shows the effect of RTK ligand HGF on "wet" AMD RPE (15-10-021) phagocytosis.
圖32顯示RTK配體BDNF對於「濕性」AMD RPE(15-10-021)吞噬之作用。 Figure 32 shows the effect of RTK ligand BDNF on phagocytosis of "wet" AMD RPE (15-10-021).
圖33顯示RTK配體GDNF對於「濕性」AMD RPE(15-10-021)吞噬之作用。 Figure 33 shows the effect of RTK ligand GDNF on "wet" AMD RPE (15-10-021) phagocytosis.
圖34顯示RTK配體BDNF對於AMD RPE吞噬之作用。 Figure 34 shows the effect of RTK ligand BDNF on AMD RPE phagocytosis.
圖35顯示RTK配體HGF對於AMD RPE(ND08333)吞噬之作用。 Figure 35 shows the effect of RTK ligand HGF on AMD RPE (ND08333) phagocytosis.
圖36顯示RTK配體GDNF對於AMD RPE(ND08333)吞噬之作用。 Figure 36 shows the effect of RTK ligand GDNF on AMD RPE (ND08333) phagocytosis.
圖37顯示RTK配體GDNF對於AMD RPE(ND08626)吞噬之作用。 Figure 37 shows the effect of RTK ligand GDNF on AMD RPE (ND08626) phagocytosis.
圖38顯示RTK配體BDNF對於AMD RPE(ND08626)吞噬之作用。 Figure 38 shows the effect of RTK ligand BDNF on AMD RPE (ND08626) phagocytosis.
圖39顯示RTK配體HGF對於AMD RPE(ND08626)吞噬之作用。 Figure 39 shows the effect of RTK ligand HGF on AMD RPE (ND08626) phagocytosis.
圖40顯示橋分子MFG-E8對於AMD RPE(SD1 L)吞噬之作用。 Figure 40 shows the effect of the bridge molecule MFG-E8 on AMD RPE (SD1 L) phagocytosis.
圖41顯示橋分子Tsp 1對於AMD RPE(SD1 L)吞噬之作用。 Figure 41 shows the effect of the bridge molecule Tsp 1 on AMD RPE (SD1 L) phagocytosis.
圖42顯示橋分子Tsp 2對於AMD RPE(SD1 L)吞噬之作用。 Figure 42 shows the effect of the bridge molecule Tsp 2 on AMD RPE (SD1 L) phagocytosis.
圖43顯示橋分子Tsp 1對於「濕性」AMD RPE(15-10-021)吞噬之作用。 Figure 43 shows the effect of the bridge molecule Tsp 1 on phagocytosis of "wet" AMD RPE (15-10-021).
圖44顯示橋分子MFG-E8對於「濕性」AMD RPE(15-10-021)吞噬之作用。 Figure 44 shows the effect of the bridge molecule MFG-E8 on phagocytosis of "wet" AMD RPE (15-10-021).
圖45顯示橋分子Tsp 2對於「濕性」AMD RPE(15-10-021)吞噬之作用。 Figure 45 shows the effect of the bridge molecule Tsp 2 on phagocytosis of "wet" AMD RPE (15-10-021).
圖46顯示橋分子MFG-E8對於AMD RPE(ND08333)吞噬之作用。 Figure 46 shows the effect of the bridge molecule MFG-E8 on AMD RPE (ND08333) phagocytosis.
圖47顯示橋分子Tsp 1對於AMD RPE(ND08333)吞噬之作用。 Figure 47 shows the effect of the bridge molecule Tsp 1 on AMD RPE (ND08333) phagocytosis.
圖48顯示橋分子Tsp 2對於AMD RPE(ND08333)吞噬之作用。 Figure 48 shows the effect of bridge molecule Tsp 2 on phagocytosis of AMD RPE (ND08333).
圖49顯示橋分子MFG-E8對於AMD RPE(ND08626)吞噬之作用。 Figure 49 shows the effect of the bridge molecule MFG-E8 on AMD RPE (ND08626) phagocytosis.
圖50顯示橋分子Tsp 1對於AMD RPE(ND08626)吞噬之作用。 Figure 50 shows the effect of bridge molecule Tsp 1 on AMD RPE (ND08626) phagocytosis.
圖51顯示橋分子Tsp 2對於AMD RPE(ND08626)吞噬之作用。 Figure 51 shows the effect of the bridge molecule Tsp 2 on phagocytosis of AMD RPE (ND08626).
本發明之其他特性及優點將由以下實施方式與實例顯見。 Other features and advantages of the present invention will be apparent from the following embodiments and examples.
本說明書中提及各種專利及其他出版物。這些出版物之各者之全部內容以引用方式併入本文中。在下列說明性實施例之詳細說明中,參照形成本說明書之一部分的隨附圖式。這些實施例係以足夠詳細之方式說明以讓熟習該項技術者能夠實行本發明,並會理解到尚可利用其他實施例,而且可在不偏離本發明之精神或範疇下作出邏輯結構、機械、電性與化學上的變化。為了避免非為所屬技術領域中具有通常知識者能夠實行本文中所述之實施例所需之細節,本說明書可能會省略某些所屬技術領域中具有通常知識者所習知之資訊。因此,以下實施方式不應理解為限制性意義,且說明性實施例之範疇由隨附申請專利範圍所限定。 Various patents and other publications are mentioned in this specification. The entire contents of each of these publications are incorporated herein by reference. In the following detailed description of the illustrative embodiments, reference is made to the accompanying drawings that form part of this specification. These embodiments are described in sufficient detail to allow those skilled in the art to implement the present invention, and will understand that other embodiments can be utilized, and logical structures and machinery can be made without departing from the spirit or scope of the present invention , Electrical and chemical changes. In order to avoid details that are not necessary for those with ordinary knowledge in the technical field to implement the embodiments described herein, this specification may omit certain information known to those with ordinary knowledge in the technical field. Therefore, the following embodiments should not be construed in a limiting sense, and the scope of the illustrative examples is defined by the scope of the accompanying patent applications.
本說明書及申請專利範圍中所使用之各種用語如以下闡述所定義且意欲闡明本發明。 The various terms used in this specification and the scope of patent application are defined as set forth below and are intended to clarify the present invention.
幹細胞(stem cell)為未分化之細胞,其係以單一細胞同時具有自我更新及分化以生產後裔細胞(progeny cell)的能力來定義,包括自我更新前驅(self-renewing progenitor)、非更新前驅(non-renewing progenitor)與最終分化細胞(terminally differentiated cell)。幹細胞之特徵亦在於其具有體外分化為多個胚層(內胚層、中胚層與外胚層)之各種細胞譜系之功能性細胞的能力,以及在移植後形成多個胚層之組織的能力,並且能夠在注入胚胞(blastocyst)後實質上促成大多數(如果不是全部)組織形成。 Stem cells are undifferentiated cells, which are defined by the ability of a single cell to simultaneously renew and differentiate to produce progeny cells, including self-renewing progenitors and non-renewing progenitors ( non-renewing progenitor) and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate into functional cells of various cell lineages of multiple germ layers (endoderm, mesoderm and ectoderm) in vitro, and the ability to form multiple germ layer tissues after transplantation The injection of blastocysts substantially contributes to the formation of most, if not all, tissues.
幹細胞依據其發展潛能分類如下:(1)全能性(totipotent);(2)多能性(pluripotent);(3)多潛能性(multipotent);(4)少能性(oligopotent);與(5)單能性(unipotent)。全能性細胞能夠形成所有胚胎與胚外細胞型。多能性細胞能夠形成所有胚胎細胞型。多潛能性細胞包括該些能夠形成細胞譜系亞群,但全部在特定組織、器官、或生理系統內之細胞(例如,造血幹細胞(HSC)可生產後裔,包括HSC(自我更新)、血液細胞限制少能性前驅、及作為血液正常組分之所有細胞型及元件(例如,血小板))。少能性細胞可以形成比多潛能性幹細胞更受限之細胞譜系亞群;及單能性細胞能夠形成單一細胞譜系(例如生精幹細胞)。 Stem cells are classified according to their developmental potential as follows: (1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and (5 ) Unipotent. Totipotent cells can form all embryonic and extraembryonic cell types. Pluripotent cells can form all embryonic cell types. Pluripotent cells include those cells that can form a subset of cell lineages, but all are within a specific tissue, organ, or physiological system (eg, hematopoietic stem cells (HSC) can produce progeny, including HSC (self-renewal), blood cell restriction The precursor of dysfunction, and all cell types and components that are normal components of blood (eg, platelets). Pluripotent cells can form a more restricted subset of cell lineages than pluripotent stem cells; and unipotent cells can form a single cell lineage (eg spermatogenic stem cells).
幹細胞亦根據其獲得來源來分類。成體幹細胞(adult stem cell)通常為多潛能性未分化細胞,其在包含多重分化細胞型之組織中發現。成體幹細胞可自我更新。在正常環境下,其亦可分化以產生其始源組織之特化細胞型,並且亦可能產生其他組織型。誘導型多能性幹細胞(iPS細胞)為轉變成多能性幹細胞之成體細胞。(Takahashi等人,Cell,2006;126(4):663-676;Takahashi等人,Cell,2007;131:1-12)。胚胎幹細胞(embryonic stem cell)為來自胚胞階段胚胎之內細胞團(inner cell mass)的多能性細胞。胎體幹細胞(fetal stem cell)為源自胎體組織或膜之幹細胞。產後幹細胞(postpartum stem cell)為多潛能性或多能性細胞,其實質上源自生產後所能取得之胚外組織,亦即胎盤及臍帶。已發現這些細胞擁有多能性幹細胞之特性特徵,包括快速增生及分化為多種細胞譜系之潛能。產後幹細胞可為血液衍生(例如該些得自臍帶血之幹細胞)或非血液衍生(例如得自臍帶及胎盤之非血液組織)。 Stem cells are also classified according to the source from which they were obtained. Adult stem cells are usually pluripotent undifferentiated cells, which are found in tissues containing multiple differentiated cell types. Adult stem cells can renew themselves. Under normal circumstances, it can also differentiate to produce a specialized cell type of its original tissue, and may also produce other tissue types. Induced pluripotent stem cells (iPS cells) are adult cells transformed into pluripotent stem cells. (Takahashi et al., Cell , 2006; 126 (4): 663-676; Takahashi et al., Cell , 2007; 131: 1-12). Embryonic stem cells are pluripotent cells derived from the inner cell mass of embryos at the embryonic cell stage. Fetal stem cells are stem cells derived from fetal tissues or membranes. Postpartum stem cells are pluripotent or pluripotent cells, which are essentially derived from extra-embryonic tissues available after production, that is, the placenta and umbilical cord. These cells have been found to possess the characteristics of pluripotent stem cells, including the potential for rapid proliferation and differentiation into multiple cell lineages. Postpartum stem cells can be blood-derived (such as those derived from cord blood) or non-blood-derived (such as non-blood tissue derived from the umbilical cord and placenta).
胚胎組織通常定義為源自胚胎(在人類係指自受精至發育約六週之期間)之組織。胎體組織係指源自胎體之組織,胎體在人類係指自發育約六週至分娩之期間。胚外組織為與胚胎或胎體相關但非源自胚胎或胎體之組織。胚外組織包括胚外膜(絨毛膜、羊膜、卵黃囊及尿囊)、臍帶及胎盤(其自身形成自絨毛膜及母體底蛻膜)。 Embryonic tissue is generally defined as tissue derived from an embryo (in humans, a period of about six weeks from fertilization to development). Carcass tissue refers to tissue derived from the carcass, which refers to the period from about six weeks of development to delivery in humans. Extra-embryonic tissue is tissue that is related to the embryo or carcass but not derived from it. Extra-embryonic tissues include the extra-embryonic membranes (chorionic membrane, amniotic membrane, yolk sac, and allantoic), umbilical cord, and placenta (which themselves form from the chorion and maternal decidua).
分化(Differentiation)為未特化(「未定向(uncommitted)」)或特化較低之細胞藉以獲得特化細胞(諸如舉例來說神經細胞或肌肉細胞)特徵的過程。已分化細胞為佔據細胞譜系內較高特化(「定向(committed)」)位置之細胞。用語定向(committed)當應用於分化過程時,係指細胞在分化路徑中已進行達到一點,在正常環境下達到該點之細胞將會持續分化為特定細胞型或細胞型亞群,並且在正常環境下無法分化為不同細胞型或回復為較低分化之細胞型。去分化(de-differentiation)係指細胞藉以回復至細胞譜系內較低特化(或定向)位置的過程。如本文中所使用,細胞之譜系(lineage)定義細胞之遺傳,即細胞係來自哪些細胞以及細胞可形成 哪些細胞。細胞之譜系將細胞放置於發育及分化之遺傳方案(hereditary scheme)內。 Differentiation is the process by which unspecified ("uncommitted") or less specialized cells acquire characteristics of specialized cells (such as, for example, nerve cells or muscle cells). Differentiated cells are cells that occupy higher specialized ("committed") positions within the cell lineage. The term "committed" when applied to the differentiation process means that the cell has reached a point in the differentiation path. Under normal circumstances, the cell that reaches this point will continue to differentiate into a specific cell type or subtype of cell type, and it is normal Under the environment, it cannot differentiate into different cell types or revert to lower differentiated cell types. De-differentiation refers to the process by which cells revert to lower specialized (or directed) positions within the cell lineage. As used herein, the lineage of a cell defines the inheritance of the cell, ie which cells the cell line comes from and which cells the cell can form. The lineage of cells places the cells in a hereditary scheme for development and differentiation.
廣義而言,前驅細胞(progenitor cell)為能夠產生較其本身更高分化之後裔,但又保有補充前驅池之能力的細胞。就此定義而言,幹細胞本身亦為前驅細胞,如同終末分化細胞之較直接前體細胞(more immediate precursor)。當提及本發明之細胞時,如以下所更詳述說明者,可使用此較廣之前驅細胞定義。狹義而言,前驅細胞通常定義為在分化路徑中作為中間者之細胞,即其係由幹細胞所形成並且在成熟細胞型或細胞型亞群之生產中作為中間者。此類型之前驅細胞通常無法自我更新。因此,如果在本文中提及此類型之細胞,將會稱其為非新生前驅細胞(non-renewing progenitor cell)或中間前驅或前體細胞(intermediate progenitor or precursor cell)。 Broadly speaking, a progenitor cell (progenitor cell) is a cell that can produce a more differentiated descendant than itself, but retains the ability to replenish the precursor pool. In this definition, stem cells themselves are also precursor cells, like more immediate precursors of terminally differentiated cells. When referring to the cells of the present invention, as described in more detail below, this broader definition of precursor cells can be used. In a narrow sense, a precursor cell is usually defined as a cell that acts as an intermediary in the differentiation pathway, that is, it is formed by stem cells and acts as an intermediary in the production of mature cell types or subpopulations of cell types. This type of prodromal cell is usually unable to renew itself. Therefore, if this type of cell is mentioned in this article, it will be referred to as a non-renewing progenitor cell or an intermediate progenitor or precursor cell.
如本文中所使用,片語「分化成眼譜系或表型(differentiates into an ocular lineage or phenotype)」係指細胞變得部分或完全定向至特定眼表型,包括但不限於視網膜及角膜幹細胞、視網膜及虹膜之色素上皮細胞、光受體、視網膜神經節及其他視神經譜系(例如,視網膜神經膠細胞、微膠細胞、星狀細胞、繆氏細胞(Mueller cell))、形成水晶體之細胞、及鞏膜、角膜、角膜緣(limbus)及結膜之上皮細胞。片語「分化成神經譜系或表型(differentiates into a neural lineage or phenotype)」係指細胞變得部分或完全定向至CNS或PNS之特定神經表型,亦即,神經元或膠細胞,後一類別包括但 不限於星狀細胞、寡樹突細胞、許旺氏細胞(Schwann cell)及微膠細胞。 As used herein, the phrase "differentiates into an ocular lineage or phenotype" refers to cells that become partially or fully directed to a specific ocular phenotype, including but not limited to retinal and corneal stem cells, Pigment epithelial cells of the retina and iris, photoreceptors, retinal ganglia, and other optic nerve lineages (eg, retinal glial cells, microglia cells, stellate cells, Mueller cells), water crystal forming cells, and Sclera, cornea, limbus (limbus) and conjunctival epithelial cells. The phrase "differentiates into a neural lineage or phenotype" refers to a specific neural phenotype in which cells become partially or completely directed to the CNS or PNS, that is, neurons or glial cells, the latter Categories include, but are not limited to, stellate cells, oligodendritic cells, Schwann cells, and micelles.
本文所例示且較佳用於本發明之細胞通常稱為產後衍生細胞(或PPDC)。其有時亦可更具體地被稱為臍衍生細胞或胎盤衍生細胞(UDC或PDC)。此外,該些細胞可被描述為幹細胞或前驅細胞(progenitor cell),後項用語係以廣義的方式來使用。用語衍生(derived)係用來指明細胞係得自其生物來源,且於體外生長或經其他方式調控(例如培養於生長培養基以擴增族群及/或生產細胞系(cell line))。臍幹細胞及胎盤幹細胞之體外調控以及本發明之臍衍生細胞及胎盤衍生細胞之獨有特性係詳述於後文中。藉由其他手段分離自產後胎盤及臍之細胞亦被認為適合用於本發明中。這些其他細胞在本文中稱為產後細胞(而非產後衍生細胞)。 The cells exemplified herein and preferably used in the present invention are generally referred to as postpartum-derived cells (or PPDC). It may sometimes be more specifically referred to as umbilical derived cells or placental derived cells (UDC or PDC). In addition, these cells may be described as stem cells or progenitor cells, and the latter term is used in a broad manner. The term derived is used to indicate that the cell line is derived from its biological source, and is grown in vitro or otherwise regulated (eg, cultured in growth medium to expand populations and / or cell lines). The in vitro regulation of umbilical stem cells and placental stem cells and the unique characteristics of the umbilical derived cells and placental derived cells of the present invention are described in detail later. Cells isolated from the postpartum placenta and umbilicus by other means are also considered suitable for use in the present invention. These other cells are referred to herein as postpartum cells (not postpartum derived cells).
使用各種用語來描述在培養物中之細胞。細胞培養物(cell culture)通常係指取自活體生物並且在受控制條件下生長(「在培養物中(in culture)」或「經培養(cultured)」)的細胞。初代細胞培養物(primary cell culture)為直接取自生物且在第一次亞培養前之細胞、組織或器官的培養物。當細胞置於生長培養基中並處於有利細胞生長及/或分裂之條件下時,其在培養物中擴增從而導致更大的細胞群。當細胞在培養物中擴增時,細胞增生之速率有時係以細胞數目倍增所需的時間量來量測。此稱為倍增時間(doubling time)。 Various terms are used to describe the cells in culture. Cell culture generally refers to cells taken from living organisms and grown under controlled conditions ("in culture" or "cultured"). Primary cell culture is a culture of cells, tissues, or organs taken directly from the organism and before the first subculture. When cells are placed in growth medium and under conditions favorable for cell growth and / or division, they expand in culture resulting in a larger cell population. When cells are expanded in culture, the rate of cell proliferation is sometimes measured by the amount of time required to double the number of cells. This is called the doubling time.
細胞系(cell line)為由初代細胞培養物之一或多個亞培養(subcultivation)所形成的細胞群。每一輪亞培養稱為一個繼代 (passage)。當細胞經過亞培養時,將它們稱為已繼代。特定之細胞群或細胞系有時會以其繼代次數來指稱或表徵。例如,繼代十次的經培養細胞群可稱為P10培養物。初代培養物(在細胞自組織分離出來後的第一次培養物)係命名為P0。在第一次亞培養後,該些細胞係描述為二次培養物(P1或繼代1)。在第二次亞培養後,該些細胞即變成三次培養物(P2或繼代2),依此類推。所屬技術領域中具有通常知識者將會理解到,在繼代期間會有多次族群倍增;因此,培養物之族群倍增次數大於其繼代次數。在繼代間隔期間的細胞擴增(即族群倍增次數)取決於許多因素,包括但不限於接種密度、基材、培養基、生長條件及繼代間隔時間。 A cell line is a cell population formed by one or more subcultivations of primary cell cultures. Each round of subculture is called a passage. When cells are subcultured, they are said to have been passaged. Certain cell populations or cell lines are sometimes referred to or characterized by their number of passages. For example, a cultured cell population subcultured ten times may be referred to as a P10 culture. The primary culture (the first culture after the cells were separated from the tissue) was named P0. After the first subculture, these cell lines were described as secondary cultures (P1 or subculture 1). After the second subculture, the cells became a three-culture (P2 or subculture 2), and so on. Those of ordinary skill in the art will understand that there will be multiple population doublings during the subculture; therefore, the number of population doublings of the culture is greater than the number of subcultures. The expansion of cells during the passage interval (ie, the number of population doublings) depends on many factors, including but not limited to seeding density, substrate, culture medium, growth conditions, and passage interval time.
用語生長培養基(Growth Medium)通常係指足以培養PPDC之培養基。詳言之,用於培養本發明之細胞的一種目前較佳培養基包含達爾伯克改良必需培養基(Dulbecco's Modified Essential Media)(在本文中亦縮寫為DMEM)。尤其較佳的是DMEM-低葡萄糖(在本文中亦稱為DMEM-LG)(Invitrogen,Carlsbad,Calif.)。DMEM-低葡萄糖較佳係補充有15%(v/v)胎牛血清(例如特級胎牛血清(defined fetal bovine serum),Hyclone,Logan Utah)、抗生素/抗黴劑(較佳為50至100個單位/毫升青黴素、50至100微克/毫升鏈黴素、及0至0.25微克/毫升兩性黴素B;Invitrogen,Carlsbad,Calif.)、及0.001%(v/v)2-巰基乙醇(Sigma,St.Louis Mo.)。如以下實例中所用,生長培養基係指具有15%胎牛血清及抗生素/抗黴劑之DMEM-低葡萄糖(當包括青黴素/鏈黴素時,其較佳分別為50U/ml 及50微克/ml;當使用青黴素/鏈黴素/兩性黴素時,其較佳分別為100U/ml、100微克/ml、及0.25微克/ml)。在一些情況下,使用不同生長培養基,或提供不同補充劑,這些通常在文中指明為生長培養基之補充劑。 The term growth medium generally refers to a medium sufficient to cultivate PPDC. In detail, one currently preferred medium for culturing the cells of the present invention includes Dulbecco's Modified Essential Media (also abbreviated as DMEM herein). Particularly preferred is DMEM-low glucose (also referred to herein as DMEM-LG) (Invitrogen, Carlsbad, Calif.). DMEM-low glucose is preferably supplemented with 15% (v / v) fetal bovine serum (such as defined fetal bovine serum (Hyclone, Logan Utah)), antibiotics / antimycotics (preferably 50 to 100 Units / ml penicillin, 50 to 100 μg / ml streptomycin, and 0 to 0.25 μg / ml amphotericin B; Invitrogen, Carlsbad, Calif.), And 0.001% (v / v) 2-mercaptoethanol (Sigma , St. Louis Mo.). As used in the following examples, the growth medium refers to DMEM-low glucose with 15% fetal bovine serum and antibiotics / antimycotics (when penicillin / streptomycin is included, it is preferably 50U / ml and 50 micrograms / ml, respectively) ; When penicillin / streptomycin / amphotericin is used, it is preferably 100 U / ml, 100 μg / ml, and 0.25 μg / ml, respectively). In some cases, different growth media are used, or different supplements are provided, which are usually indicated in the text as supplements for growth media.
條件培養基(conditioned medium)為其中特定細胞或細胞群係經培養且隨後移除之培養基。當細胞培養於培養基中時,其可分泌可提供營養支持給其他細胞之細胞因子。此類營養因子包括但不限於荷爾蒙、細胞介素、細胞外基質(ECM)、蛋白質、囊泡、抗體、及顆粒。含有細胞因子之培養基為條件培養基。 A conditioned medium is a medium in which specific cells or cell population lines are cultured and then removed. When cells are cultured in a medium, they can secrete cytokines that can provide nutritional support to other cells. Such nutritional factors include, but are not limited to hormones, interleukins, extracellular matrix (ECM), proteins, vesicles, antibodies, and particles. The medium containing cytokines is conditioned medium.
通常,營養因子(trophic factor)係定義為促進細胞生存、生長、分化、增生及/或成熟之物質,或刺激細胞提高活性之物質。細胞之間經由營養因子的交互作用可發生在不同類型的細胞之間。藉助營養因子之細胞交互作用見於基本上所有的細胞型中,且為神經細胞型之通訊所尤其顯著的手段。營養因子亦可以自泌(autocrine)之方式作用,亦即,細胞可生產影響其自身生存、生長、分化、增生及/或成熟之營養因子。 Generally, trophic factors are defined as substances that promote cell survival, growth, differentiation, proliferation, and / or maturation, or substances that stimulate cell activity. The interaction between cells via trophic factors can occur between different types of cells. Cell interactions with the help of trophic factors are found in essentially all cell types, and are a particularly significant means of communication for nerve cell types. Nutritional factors can also act in an autocrine manner, that is, cells can produce nutritional factors that affect their own survival, growth, differentiation, proliferation, and / or maturation.
當指稱經培養之脊椎動物細胞時,用語衰老(senescence)(複製衰老(replicative senescence)或細胞衰老(cellular senescence)亦同)係指可歸因於有限細胞培養之性質;亦即,它們無能力生長超越有限的族群倍增次數(有時稱為海富利克限度(Hayflick's limit))。儘管細胞衰老最先係使用纖維母細胞樣細胞(fibroblast-like cell)來描述,但是可在培養物中成功生長之大部分正 常人類細胞型均經歷細胞衰老。不同細胞型之體外壽命不同,但是最大壽命通常少於100次族群倍增(此為培養物中之所有細胞變衰老且因此使培養物無法分裂之倍增次數)。衰老並非取決於時序時間,而是由培養物所經歷之細胞分裂、或族群倍增之次數所量測。 When referring to cultured vertebrate cells, the term senescence (replicative senescence or cellular senescence is also the same) refers to properties attributable to limited cell culture; that is, they are incapable of Growth exceeds the limited number of population doublings (sometimes called Hayflick's limit). Although cell senescence was first described using fibroblast-like cells, most normal human cell types that can successfully grow in culture undergo cell senescence. Different cell types have different life spans in vitro, but the maximum life span is usually less than 100 population doublings (this is the number of doublings in which all cells in the culture become senescent and therefore make the culture unable to divide). Aging does not depend on time sequence, but is measured by the number of cell divisions or population doublings experienced by the culture.
用語眼(ocular)、眼科(ophthalmic)及視(optic)在本文中可互換使用以定義「眼睛的、或有關眼睛、或與眼睛相關(of,or about,or related to the eye)」。用語眼變性病狀(ocular degenerative condition)(或病症(disorder))為包括性用語,涵蓋涉及細胞損傷、變性或損失之眼睛(包括眼睛與腦之間的神經連接)的急性及慢性病狀、病症或疾病。眼變性病狀可與年齡有關,或可因受傷或創傷所致,或可與特定疾病或病症有關。急性眼變性病狀包括但不限於與細胞死亡相關之病狀或影響眼睛的損害,包括由以下引起之病狀:腦血管機能不全、局灶性或瀰漫性腦創傷、瀰漫性腦損傷、傳染性或發炎性眼睛病狀、視網膜裂開或脫附、眼內病灶(挫傷穿透、壓迫、撕裂)或其他身體受傷(例如,物理或化學灼傷)。慢性眼變性病狀(包括進行性病狀)包括但不限於視網膜病變及其他視網膜/黃斑病症,諸如色素性視網膜炎(RP)、年齡相關性黃斑變性(AMD)、脈絡膜新生血管膜(CNVM);視網膜病變,諸如糖尿病視網膜病變、閉塞性視網膜病變、鐮狀細胞視網膜病變及高血壓視網膜病變、中央視網膜靜脈阻塞、頸動脈狹窄、視神經病變,諸如青光眼及相關症候群;水晶體及外眼之病症例如角膜緣幹細胞缺乏(LSCD),亦稱為角膜緣上皮細胞缺乏(LECD),諸如發生於化學或熱受傷中、史蒂 芬強森症候群症候群(Steven-Johnson syndrome)、隱形眼鏡誘導之角膜病變、眼瘢痕性類天皰瘡、先天無虹膜或外胚層發育不良疾病、及多發性內分泌缺乏相關角膜炎。 The terms ocular, ophthalmic, and optic are used interchangeably herein to define "of, or about, or related to the eye." The term ocular degenerative condition (or disorder) is an inclusive term that covers acute and chronic conditions and disorders involving the eyes (including the neural connection between the eye and the brain) of cell damage, degeneration or loss Or disease. Eye degeneration symptoms may be related to age, or may be caused by injury or trauma, or may be related to a specific disease or condition. Acute ocular degenerative conditions include but are not limited to conditions related to cell death or damage to the eye, including conditions caused by: cerebrovascular insufficiency, focal or diffuse brain trauma, diffuse brain injury, infection Sexual or inflammatory eye conditions, split or detached retina, intraocular lesions (contusion penetration, compression, tearing) or other physical injuries (eg, physical or chemical burns). Chronic ocular degeneration conditions (including progressive conditions) include but are not limited to retinopathy and other retinal / macular diseases, such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), choroidal neovascular membrane (CNVM); Retinopathy, such as diabetic retinopathy, occlusive retinopathy, sickle cell retinopathy and hypertensive retinopathy, central retinal vein occlusion, carotid artery stenosis, optic neuropathy, such as glaucoma and related syndromes; water lens and external eye disorders such as cornea Limbry stem cell deficiency (LSCD), also known as limbal epithelial cell deficiency (LECD), such as occurs in chemical or thermal injuries, Steven-Johnson syndrome, contact lens-induced corneal lesions, eye scarring Pemphigoid, congenital absence of iris or ectodermal dysplasia, and multiple endocrine deficiency related keratitis.
用語治療眼變性病狀(treating an ocular degenerative condition)或眼變性病狀之治療(treatment of an ocular degenerative condition)係指緩解如本文所定義之眼變性病狀之作用,或延緩、暫停或逆轉眼變性病狀之進展,或延緩或預防眼變性病狀之發生。 The term treatment of an ocular degenerative condition or treatment of an ocular degenerative condition refers to the effect of relieving an ocular degenerative condition as defined herein, or delaying, suspending or reversing the eye The progress of degenerative symptoms, or delay or prevent the occurrence of ocular degenerative symptoms.
用語有效量(effective amount)係指試劑或醫藥組成物諸如生長因子、分化劑、營養因子、細胞群或其他藥劑之濃度或量,該濃度或量可有效產生預期結果,包括如本文所述之體外或體內之細胞生長及/或分化,或治療眼變性病狀。關於生長因子,有效量可在約1奈克/毫升至約1微克/毫升之範圍內。關於向患者體內投予之PPDC,有效量可在少至數百或更少、至多達數百萬或更多之範圍內。在特定實施例中,有效量可在103至1111個細胞之範圍內,更特定言之為至少約104個細胞。應理解待投予之細胞數目將取決於待治療病症之細節,包括但不限於待治療之大小或總體積/表面積,以及投予位點與待治療區域之位置之靠近性,以及醫藥生物學家熟悉的其他因素而變化。 The term effective amount refers to the concentration or amount of an agent or pharmaceutical composition such as growth factors, differentiation agents, trophic factors, cell populations, or other agents that is effective to produce the desired result, including as described herein Cell growth and / or differentiation in vitro or in vivo, or treatment of ocular degeneration. With regard to growth factors, the effective amount may range from about 1 ng / ml to about 1 μg / ml. With regard to PPDC administered to a patient, the effective amount may range from as few as hundreds or less to as many as millions or more. In a particular embodiment, an effective amount can range from 10 3 to 11 11 cells, the more specific words of at least about 104 cells. It should be understood that the number of cells to be administered will depend on the details of the condition to be treated, including but not limited to the size or total volume / surface area to be treated, the proximity of the administration site to the area to be treated, and medical biology The home is familiar with other factors.
用語有效期間(effective period)(或時間(time))及有效條件(effective condition)係指對於劑或醫藥組成物而言,為達成其預期結果所必需或較佳的時間期間或其他可控條件(例如,體外方法之溫度、濕度)。 The terms effective period (or time) and effective condition refer to the period of time or other controllable conditions necessary or better for the agent or pharmaceutical composition to achieve its expected result (For example, the temperature and humidity of the in vitro method).
用語患者(patient)或個體(subject)係指用本文所述之醫藥組成物或根據本文所述之方法治療之動物,包括哺乳動物,較佳為人類。 The terms patient or subject refer to animals treated with the pharmaceutical composition described herein or according to the methods described herein, including mammals, preferably humans.
用語醫藥上可接受之載劑(pharmaceutically acceptable carrier)(或介質(medium))可與用語生物可相容之載劑或介質(biologically compatible carrier or medium)互換使用,係指試劑、細胞、化合物、材料、組成物、及/或劑型,其不僅可與待治療性投予之細胞及其他藥劑相容,而且在合理的醫學判斷之範疇內適用於與人類及動物之組織接觸,而無過度毒性、刺激、過敏反應、或符合合理的利益/風險比的其他併發症。 The term pharmaceutically acceptable carrier (or medium) can be used interchangeably with the term biologically compatible carrier or medium, which refers to reagents, cells, compounds, Materials, compositions, and / or dosage forms are not only compatible with the cells and other agents to be administered therapeutically, but also suitable for contact with human and animal tissues without excessive toxicity within the scope of reasonable medical judgment , Irritation, allergic reactions, or other complications in a reasonable benefit / risk ratio.
在本文中使用數個關於細胞置換療法(cell replacement therapy)之用語。用語自體轉移(autologous transfer)、自體移植(autologous transplantation)、自體移接(autograft)及類似用語係指其中細胞捐贈者亦為細胞置換療法之接受者之治療。用語同種異體轉移(allogeneic transfer)、同種異體移植(allogeneic transplantation)、同種異體移接(allograft)及類似用語係指其中細胞捐贈者與細胞置換療法之接受者為相同物種,但非相同個體之治療。捐贈者細胞與接受者組織相容性匹配之細胞轉移有時稱為同基因轉移(syngeneic transfer)。用語異種轉移(xenogeneic transfer)、異種移植(xenogeneic transplantation)、異種移接(xenograft)及類似用語係指其中細胞捐贈者與細胞置換療法之接受者為不同物種之治療。 如本文中所使用之移植係指將自體、或同種異體捐贈者細胞置換療法引入至接受者。 Several terms related to cell replacement therapy are used herein. The terms autologous transfer, autologous transplantation, autograft and similar terms refer to the treatment in which the cell donor is also the recipient of cell replacement therapy. The terms allogeneic transfer (allogeneic transfer), allogeneic transplantation (allogeneic transplantation), allograft (allograft) and similar terms refer to the treatment in which the cell donor and the recipient of the cell replacement therapy are the same species, but not the same individual . Donor cells and recipients' cell compatibility matching cell transfer is sometimes referred to as syngeneic transfer (syngeneic transfer). The terms xenogeneic transfer, xenogeneic transplantation, xenograft, and similar terms refer to the treatment in which the cell donor and the recipient of cell replacement therapy are different species. Transplantation as used herein refers to the introduction of autologous, or allogeneic donor cell replacement therapy to the recipient.
如本文中所使用,用語「約(about)」在指稱一可測量的值(例如數量、持續時間及類似者)時,意欲涵蓋在指定值之±20%與±0.1%之間、較佳為±20%或±10%、更佳為±5%、甚至更佳為±1%、且仍更佳為±0.1%的變異,若此類變異為適合執行所揭露之方法者。 As used herein, the term "about" when referring to a measurable value (such as quantity, duration, and the like) is intended to cover between ± 20% and ± 0.1% of the specified value, preferably Variations of ± 20% or ± 10%, more preferably ± 5%, even better ± 1%, and still more preferably ± 0.1%, if such variations are suitable for performing the disclosed method.
眼變性病狀涵蓋成因分歧之急性、慢性及進行性病症及疾病,共同特徵為特定或易受傷害之眼細胞群的功能異常或損失。此共同性使能夠開發用於修復或再生易受傷害、受損或失去之眼組織之類似治療方式,其中之一為基於細胞之療法。眼變性病狀之細胞療法的開發受限於相對較少類型的幹細胞或前驅細胞,包括眼衍生幹細胞本身(例如,視網膜及角膜幹細胞)、胚胎幹細胞及少數類型的成體幹細胞或前驅細胞(例如,神經、黏膜上皮及骨髓幹細胞)。分離自產後臍帶及胎盤之細胞已被識別為適用於此目的之前驅細胞之顯著新來源。(US 2005-0037491及US 2010-0272803)此外,由分離自產後胎盤及臍帶組織之細胞所產生之條件培養基提供另一種用於治療眼變性病狀之新來源。因此,在本文所述之各種實施例中,本發明之特徵在於用於修復及再生眼組織之方法及組成物(包括醫藥組成物),該方法及組成物使用來自分離自產後組織之前 驅細胞及細胞群之條件培養基。本發明適用於眼變性病狀,但預期尤其適用於一些難以治療或治癒或無可得治療或療法之眼病症。這些眼病症包括但不限於年齡相關性黃斑變性、色素性視網膜炎、糖尿病性及其他視網膜病變。 The ocular degeneration pathology covers acute, chronic, and progressive disorders and diseases with different causes. The common feature is the abnormal or loss of function of specific or vulnerable eye cell populations. This commonality enables the development of similar treatments for repairing or regenerating vulnerable, damaged or lost eye tissue, one of which is cell-based therapy. The development of cell therapies for ocular degenerative diseases is limited to relatively few types of stem cells or precursor cells, including eye-derived stem cells themselves (eg, retinal and corneal stem cells), embryonic stem cells, and a few types of adult stem cells or precursor cells (eg , Nerve, mucosal epithelium and bone marrow stem cells). Cells isolated from the postpartum umbilical cord and placenta have been identified as a significant new source of precursor cells suitable for this purpose. (US 2005-0037491 and US 2010-0272803) In addition, conditioned medium produced from cells isolated from postpartum placenta and umbilical cord tissue provides another new source for the treatment of ocular degeneration. Therefore, in the various embodiments described herein, the present invention is characterized by a method and composition (including a pharmaceutical composition) for repairing and regenerating eye tissue, which uses precursor cells isolated from postpartum tissue And conditioned medium for cell populations. The present invention is applicable to ocular degenerative conditions, but it is expected to be particularly applicable to some ocular disorders that are difficult to treat or cure or have no available treatment or therapy. These eye conditions include, but are not limited to, age-related macular degeneration, retinitis pigmentosa, diabetic, and other retinopathy.
預期衍生自前驅細胞(諸如根據所屬技術領域中已知之任何方法分離自產後臍帶或胎盤之細胞)之條件培養基適合用於本發明中。然而,在一實施例中,本發明使用衍生自如上文所定義之臍帶組織衍生細胞(hUTC)或胎盤組織衍生細胞(PDC)之條件培養基,該等細胞係衍生自較佳地根據下文所述之方法使之實質上不含血液之臍帶組織或胎盤。hUTC或PDC能夠在培養物中擴增且具有分化成其他表型之細胞的潛能。某些實施例之特徵在於由此類前驅細胞所製備之條件培養基、包含條件培養基之組成物、及使用組成物諸如醫藥組成物治療急性或慢性眼變性病狀患者之方法。本發明之產後衍生細胞係經其在培養物中之生長性質、其細胞表面標記、其基因表現、其生產某些生化營養因子之能力、及其免疫性質表徵。衍生自產後衍生細胞之條件培養基係經該些細胞所分泌之營養因子及橋分子表徵。 It is expected that conditioned medium derived from precursor cells, such as cells isolated from the postpartum umbilical cord or placenta according to any method known in the art, is suitable for use in the present invention. However, in one embodiment, the present invention uses a conditioned medium derived from umbilical cord tissue-derived cells (hUTC) or placental tissue-derived cells (PDC) as defined above, these cell lines being derived preferably from the following The method of making it substantially free of umbilical cord tissue or placenta without blood. hUTC or PDC can expand in culture and have the potential to differentiate into cells of other phenotypes. Certain embodiments are characterized by a conditioned medium prepared from such precursor cells, a composition containing the conditioned medium, and a method of using a composition such as a pharmaceutical composition to treat patients with acute or chronic ocular degenerative diseases. The postpartum-derived cells of the present invention are characterized by their growth properties in culture, their cell surface markers, their gene expression, their ability to produce certain biochemical trophic factors, and their immune properties. The conditioned medium derived from postpartum-derived cells is characterized by trophic factors and bridge molecules secreted by these cells.
用於本發明之組成物及方法中之細胞、細胞群及包含細胞溶解產物、條件培養基及其類似物之製劑係描述於本文,且詳細描述於美國專利第7,524,489號、及第7,510,873號、及美國公開 申請案第2005/0058634號中,其皆以引用方式併入本文中。根據該些方法,將哺乳動物臍帶及胎盤在足月或不足月懷孕結束時或結束後不久例如在排出胎衣(after-birth)後回收。產後組織可在無菌容器例如燒瓶、燒杯、培養皿或袋中自生產地運輸至實驗室。該容器可具有溶液或培養基,包括但不限於鹽溶液如達爾伯克改良伊格爾培養基(Dulbecco’s Modified Eagle's Medium,DMEM)或磷酸鹽緩衝液(PBS),或者任何用來運輸用於移植之器官的溶液如威斯康辛大學溶液(University of Wisconsin solution)或全氟化化合物溶液(perfluorochemical solution)。可將一或多種抗生素及/或抗黴劑(例如但不限於青黴素(penicillin)、鏈黴素(streptomycin)、兩性黴素B(amphotericin B)、建它黴素(gentamicin)與制黴菌素(nystatin))加至該培養基或緩衝液。產後組織可用抗凝劑溶液例如含肝素溶液來潤洗。較佳者為使該組織在萃取PPDC前保持在約4至10℃下。更佳者為使該組織在萃取PPDC前未經冷凍。 Cells, cell populations, and formulations containing cell lysates, conditioned media, and the like used in the compositions and methods of the present invention are described herein, and are described in detail in U.S. Patent Nos. 7,524,489, and 7,510,873, and In US Published Application No. 2005/0058634, all of which are incorporated herein by reference. According to these methods, the mammalian umbilical cord and placenta are recovered at the end of a full-term or short-term pregnancy or shortly after the end, for example, after the birth-birth is discharged. Postpartum tissues can be transported from the production site to the laboratory in sterile containers such as flasks, beakers, petri dishes or bags. The container may have a solution or culture medium, including but not limited to a saline solution such as Dulbecco's Modified Eagle's Medium (DMEM) or phosphate buffered saline (PBS), or any organ used for transportation for transplantation Such as the University of Wisconsin solution (University of Wisconsin solution) or perfluorochemical solution (perfluorochemical solution). One or more antibiotics and / or antimycotics (such as but not limited to penicillin, streptomycin, amphotericin B), gentamicin and nystatin ( nystatin)) to this medium or buffer. Postpartum tissues can be rinsed with anticoagulant solutions such as heparin-containing solutions. Preferably, the tissue is kept at about 4 to 10 ° C before PPDC extraction. Even better is that the tissue is not frozen before PPDC extraction.
PPDC之分離較佳為在無菌環境中進行。臍帶可藉由該項技術領域中習知之手段來與胎盤分開。或者,臍帶與胎盤未經分開即可使用。較佳為在分離PPDC前自產後組織移除血液與碎屑。例如,產後組織可用緩衝液來清洗,諸如但不限於磷酸鹽緩衝液。清洗緩衝液亦可包含一或多種抗黴劑及/或抗生素,諸如但不限於青黴素、鏈黴素、兩性黴素B、建它黴素及制黴菌素。 The separation of PPDC is preferably carried out in a sterile environment. The umbilical cord can be separated from the placenta by means known in the art. Alternatively, the umbilical cord and placenta can be used without being separated. It is preferable to remove blood and debris from postpartum tissues before separating PPDC. For example, postpartum tissues can be washed with buffers, such as but not limited to phosphate buffers. The washing buffer may also contain one or more anti-fungal agents and / or antibiotics, such as but not limited to penicillin, streptomycin, amphotericin B, itanomycin and nystatin.
包含完整胎盤或臍帶、或其片段或區段之產後組織係藉由機械力(切碎或剪切力)來崩解。在目前較佳實施例中,分離 程序亦利用酶消化法。許多種酶在該項技術領域中係習知可用來自複雜組織基質中分離出個別細胞以利於在培養物中生長。此類酶為市售可得,範圍從弱消化性(例如去氧核糖核酸酶及中性蛋白酶分散酶(dispase))到強消化性(例如木瓜酶及胰蛋白酶)皆有。相容於本文中之酶的非詳盡無遺清單包括黏液分解酶活性物、金屬蛋白酶(metalloprotease)、中性蛋白酶、絲胺酸蛋白酶(例如胰蛋白酶、胰凝乳蛋白酶或彈性蛋白酶)與去氧核糖核酸酶。目前較佳者為選自金屬蛋白酶、中性蛋白酶與黏液分解活性物之酶活性物。例如,膠原蛋白酶已知可用來自組織分離出各種細胞。去氧核糖核酸酶可消化單股DNA並且可使分離期間的細胞結塊(cell clumping)降至最低。較佳方法涉及用例如膠原蛋白酶及分散酶、或膠原蛋白酶、分散酶、及玻尿酸酶進行酶處理,且提供此類方法,其中在某些較佳實施例中,膠原蛋白酶與中性蛋白酶分散酶之混合物係用於解離步驟中。更佳者為該些於至少一種來自溶組織芽胞梭菌(Clostridium histolyticum)之膠原蛋白酶以及蛋白酶活性物、分散酶與嗜熱菌蛋白酶中之任一者存在下來進行消化之方法。再佳者為同時使用膠原蛋白酶與分散酶酶活性物來進行消化之方法。同樣較佳者為包括除了膠原蛋白酶與分散酶活性物外使用玻尿酸酶活性物來進行消化之方法。熟悉該項技藝人士將會瞭解到,許多種此類酶處理在該項技術領域中皆為習知可用於自各種組織來源分離出細胞。舉例而言,酶組合之LIBERASETM Blendzyme 3(Roche)系列適合用於本發明方法中。其他酶來源為習知者,並且熟悉該項技藝人士亦可直接自此類 酶之天然來源獲得這些酶。熟悉該項技藝人士亦擁有充足知識,能夠針對新的或其他酶或酶組合在用於分離本發明之細胞方面評估其實用性。較佳酶處理費時0.5、1、1.5或2小時或更久。在其他較佳實施例中,組織在解離步驟之酶處理期間係培養於37℃下。 Postpartum tissues that contain intact placenta or umbilical cord, or fragments or sections thereof are disintegrated by mechanical force (shredding or shearing force). In the presently preferred embodiment, the separation procedure also uses enzyme digestion. Many enzymes are known in the art to isolate individual cells from complex tissue matrices to facilitate growth in culture. Such enzymes are commercially available and range from weak digestibility (such as deoxyribonuclease and neutral protease dispase) to strong digestibility (such as papain and trypsin). A non-exhaustive list of enzymes compatible with this article includes mucolytic enzyme activity, metalloprotease, neutral protease, serine protease (e.g. trypsin, chymotrypsin or elastase) and deoxyribose Nuclease. Currently preferred are enzyme actives selected from metalloproteinases, neutral proteases and mucolytic activity. For example, collagenase is known to isolate various cells from tissues. Deoxyribonuclease can digest a single strand of DNA and can minimize cell clumping during separation. Preferred methods involve enzymatic treatment with, for example, collagenase and dispase, or collagenase, dispase, and hyaluronidase, and such methods are provided, wherein in certain preferred embodiments, collagenase and neutral protease dispase The mixture is used in the dissociation step. More preferably, it is a method of digesting in the presence of at least one of collagenase from Clostridium histolyticum and protease actives, dispase and thermolysin. Even better is a method of digestion using collagenase and dispase enzyme actives at the same time. It is also preferable to include a method of digesting with hyaluronidase actives in addition to collagenase and dispase actives. Those skilled in the art will understand that many such enzyme treatments are well known in the art and can be used to isolate cells from various tissue sources. For example, the LIBERASE ™ Blendzyme 3 (Roche) series of enzyme combinations is suitable for use in the method of the present invention. Other sources of enzymes are known, and those skilled in the art can also obtain these enzymes directly from the natural sources of such enzymes. Those skilled in the art also have sufficient knowledge to evaluate the utility of new or other enzymes or enzyme combinations in the isolation of cells of the invention. The preferred enzyme treatment takes 0.5, 1, 1.5 or 2 hours or more. In other preferred embodiments, the tissue is cultured at 37 ° C during the enzyme treatment in the dissociation step.
在本發明之一些實施例中,產後組織係分開為包含各種組織態樣舉例而言例如胎盤之新生兒、新生兒/母體及母體態樣的區段。分開之區段接著根據本文中所述之方法藉由機械及/或酶解離來解離。新生兒或母體譜系之細胞可藉由該項技術領域中習知的任何手段來識別,例如藉由核型分析(karyotype analysis)或Y染色體之原位雜交。 In some embodiments of the invention, the postpartum tissue is divided into sections containing various tissue morphologies such as, for example, neonates, placenta / maternal and maternal morphology of the placenta. The separated sections are then dissociated by mechanical and / or enzymatic dissociation according to the methods described herein. The cells of the newborn or maternal lineage can be identified by any means known in the art, such as by karyotype analysis or in situ hybridization of the Y chromosome.
經分離之細胞或PPDC自其所生長出之產後組織可用來起始或接種細胞培養物。將經分離之細胞轉移至無菌組織培養容器,並且容器未經塗布或經細胞外基質或配體如層黏蛋白、膠原蛋白(原生、變性或交聯)、明膠、纖連蛋白素與其他細胞外基質蛋白質塗布。PPDC係於任何能夠維持該等細胞生長之培養基中培養,諸如但不限於DMEM(高或低葡萄糖)、進階DMEM、DMEM/MCDB 201、伊格爾基礎培養基(Eagle’s basal medium)、哈姆F10培養基(Ham's F-10 medium,F10)、哈姆F-12培養基、伊思考夫改良達爾伯克培養基(Iscove’s modified Dulbecco’s medium)、間葉幹細胞生長培養基(Mesenchymal Stem Cell Growth Medium,MSCGM)、DMEM/F12、RPMI 1640及cellgro FREETM。培養基可補充一或多種組分,包括例如胎牛血清(FBS)(較佳為約2-15%(v/v));馬血清 (ES);人血清(HS);β-巰基乙醇(beta-mercaptoethanol,BME或2-ME),較佳為約0.001%(v/v));一或多種生長因子,例如血小板衍生生長因子(PDGF)、表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、血管內皮生長因子(VEGF)、類胰島素生長因子-1(IGF-1)、白血球抑制因子(LIF)及紅血球生成素;胺基酸,包括L-纈胺酸;以及一或多種抗生素及/或抗黴劑以控制微生物污染,例如青黴素G、硫酸鏈黴素、兩性黴素B、建它黴素及制黴菌素,不論單獨或組合使用。培養基較佳包含生長培養基(DMEM-低葡萄糖、血清、BME、及抗生素)。 The isolated cells or the postpartum tissue from which PPDC is grown can be used to initiate or inoculate cell cultures. Transfer the separated cells to a sterile tissue culture container, and the container is not coated or extracellular matrix or ligands such as laminin, collagen (native, denatured or cross-linked), gelatin, fibronectin and other cells Outer matrix protein coating. PPDC is cultured in any medium that can maintain the growth of these cells, such as but not limited to DMEM (high or low glucose), advanced DMEM, DMEM / MCDB 201, Eagle's basal medium, Ham F10 Medium (Ham's F-10 medium, F10), Ham F-12 medium, Iscove's modified Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM), DMEM / F12, RPMI 1640 and cellgro FREE TM . The culture medium can be supplemented with one or more components, including, for example, fetal bovine serum (FBS) (preferably about 2-15% (v / v)); horse serum (ES); human serum (HS); β-mercaptoethanol ( beta-mercaptoethanol, BME or 2-ME), preferably about 0.001% (v / v)); one or more growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth Factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), leukocyte inhibitory factor (LIF) and erythropoietin; amino acids, including L-valine; and one or A variety of antibiotics and / or anti-fungal agents to control microbial contamination, such as penicillin G, streptomycin sulfate, amphotericin B, itanomycin and nystatin, whether used alone or in combination. The culture medium preferably contains growth medium (DMEM-low glucose, serum, BME, and antibiotics).
細胞係以允許細胞生長之密度接種於培養容器中。在一較佳實施例中,細胞在約0至約5體積百分比的CO2在空氣中培養。在一些較佳實施例中,細胞在約2至約25百分比的O2在空氣中、較佳在約5至約20百分比的O2在空氣中培養。細胞較佳係在約25至約40℃下培養,且更佳係在37℃下培養。細胞較佳係在培養箱中培養。培養容器中的培養基可為靜止或經攪拌,例如使用生物反應器。較佳為使PPDC在低氧化應力(oxidative stress)下生長(例如添加麩胱甘肽、維生素C、過氧化氫酶、維生素E、N-乙醯半胱胺酸)。如本文所用之「低氧化應力(Low oxidative stress)」係指對於所培養細胞無自由基傷害或最低自由基傷害之條件。 The cell line is seeded in the culture vessel at a density that allows the cells to grow. In a preferred embodiment, the cells are cultured in air at about 0 to about 5 volume percent CO 2 . In some preferred embodiments, the cells are cultured at about 2 to about 25 percent O 2 in air, preferably at about 5 to about 20 percent O 2 in air. The preferred cell line is cultured at about 25 to about 40 ° C, and the more preferred line is cultured at 37 ° C. The cells are preferably cultured in an incubator. The culture medium in the culture vessel may be stationary or stirred, for example, using a bioreactor. Preferably, the PPDC is grown under low oxidative stress (for example, adding glutathione, vitamin C, catalase, vitamin E, N-acetylcysteine). As used herein, "low oxidative stress" refers to the condition of no or minimum free radical damage to the cultured cells.
用於選擇最適當培養基、培養基製備與細胞培養技術之方法在該項技術領域為熟知者,並且係描述於各種不同來源中,包括Doyle等人,(編),1995,Cell &TISSUE CULTURE: LABORATORY PROCEDURES,John Wiley & Sons,Chichester;與Ho及Wang(編),1991,ANIMAL CELL BIOREACTORS,Butterworth-Heinemann,Boston,上述皆以引用方式併入本文中。 Methods for selecting the most appropriate medium, medium preparation, and cell culture technology are well known in the art, and are described in various sources, including Doyle et al. (Eds.), 1995, Cell & TISSUE CULTURE: LABORATORY PROCEDURES , John Wiley & Sons, Chichester; and Ho and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS, Butterworth-Heinemann, Boston, all of which are incorporated herein by reference.
在培養分離之細胞或組織片段一段足夠時間後,PPDC將會生長出來,此係因自產後組織遷移出來或因細胞分裂所致,或者兩者皆是。在本發明的一些實施例中,PPDC係經繼代或移除至含有與初始使用之培養基相同或不同類型之新鮮培養基的分開培養容器中,其中細胞群可經有絲分裂擴增。本發明之細胞可在繼代0與衰老之間的任何時點使用。較佳為將細胞繼代約3與約25次之間,更佳為繼代約4至約12次,並且較佳為繼代10或11次。可執行選殖(Cloning)及/或次選殖(subcloning)以確認已分離出細胞的殖株族群(clonal population)。 After culturing the separated cells or tissue fragments for a sufficient period of time, PPDC will grow out, which is caused by tissue migration from postpartum or cell division, or both. In some embodiments of the invention, the PPDC is subcultured or removed to a separate culture vessel containing fresh medium of the same or different type as the medium initially used, where the cell population can be expanded by mitosis. The cells of the present invention can be used at any point between passage 0 and senescence. Preferably, the cells are passaged between about 3 and about 25 times, more preferably about 4 to about 12 times, and more preferably 10 or 11 times. Cloning and / or subcloning can be performed to confirm the clonal population of isolated cells.
在本發明之一些態樣中,存在於產後組織中的不同細胞型係經離拆(fractionated)為亞群,而PPDC可自該些亞群分離出來。此可使用細胞分離標準技術來達成,包括但不限於進行酶處理以將產後組織解離為其組分細胞,接著選殖及選擇特定細胞型,例如但不限於根據形態及/或生化標記來選擇;選擇性生長所欲之細胞(正向選擇)、選擇性破壞非所欲之細胞(負向選擇);根據混合族群中之差別細胞可凝集性來進行分離,例如使用黃豆凝集素;冷凍-解凍程序;在混合族群中之細胞的差別貼附性;過濾;傳統與區帶(zonal)離心;離心淘析(對流離心(counter-streaming centrifugation));單元重力分離(unit gravity separation);逆流分配 (countercurrent distribution);電泳;及螢光活化細胞分選(FACS)。有關殖株選擇及細胞分離技術之綜述,參見Freshney,1994,CULTURE OF ANIMAL CELLS:A MANUAL OF BASIC TECHNIQUES,第3版,Wiley-Liss,Inc.,New York,其以引用方式併入本文。 In some aspects of the invention, the different cell types present in the postpartum tissue are fractionated into subpopulations from which PPDCs can be separated. This can be achieved using standard cell separation techniques, including but not limited to enzymatic treatment to dissociate postpartum tissue into its component cells, followed by colonization and selection of specific cell types, such as but not limited to selection based on morphology and / or biochemical markers ; Selective growth of desired cells (positive selection), selective destruction of non-desired cells (negative selection); separation according to the agglutinability of differential cells in mixed populations, for example using soybean lectin; freezing- Thawing procedures; differential adherence of cells in mixed populations; filtration; traditional and zonal centrifugation; centrifugal elutriation (counter-streaming centrifugation); unit gravity separation; countercurrent Countercurrent distribution; electrophoresis; and fluorescence activated cell sorting (FACS). For a review of plant selection and cell isolation techniques, see Freshney, 1994, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, 3rd edition, Wiley-Liss, Inc., New York, which is incorporated herein by reference.
視需要更換培養基,例如使用例如吸量管小心將培養基自皿吸出,然後以新鮮培養基補足。持續培養直到皿中累積足夠細胞數目或密度。可將原始外植之組織區段取出,然後使用標準技術或使用細胞刮勺來讓剩餘細胞進行胰蛋白酶消化(trypsinized)。在胰蛋白酶消化後,將細胞收集、移至新鮮培養基然後如上述培養。在一些實施例中,培養基在胰蛋白酶消化後約24小時更換至少一次以移除任何飄浮細胞。培養物中剩餘之細胞被視為是PPDC。 If necessary, replace the culture medium, for example, carefully aspirate the medium from the dish using, for example, a pipette, and then make up with fresh medium. Continue culturing until sufficient cell number or density has accumulated in the dish. The original explanted tissue sections can be removed and then trypsinized using standard techniques or using a cell spatula. After trypsin digestion, the cells are collected, transferred to fresh medium and then cultured as described above. In some embodiments, the medium is changed at least once about 24 hours after trypsin digestion to remove any floating cells. The remaining cells in the culture are considered PPDC.
PPDC可經凍存。因此,在以下更詳細描述之較佳實施例中,用於自體轉移之PPDC(無論是用於母親或嬰兒)可衍生自在嬰兒出生後之適當產後組織,接著經過凍存以使得之後需要移植該些細胞時可供使用。 PPDC can be stored frozen. Therefore, in the preferred embodiment described in more detail below, PPDCs used for self-transfer (whether for mothers or infants) can be derived from appropriate postpartum tissues after the infant is born, and then cryopreserved so that subsequent transplantation is required These cells are available.
本發明之前驅細胞諸如PPDC可藉由下列加以表徵:例如生長特徵(例如族群倍增能力、倍增時間、到達衰老之繼代數)、核型分析(例如正常核型;母體或新生兒譜系)、流動式細胞測量術(例如FACS分析)、免疫組織化學法及/或免疫細胞化學法(例如 偵測表位)、基因表現概況(例如基因晶片陣列分析;聚合酶連鎖反應(例如反轉錄酶PCR、即時PCR及傳統PCR))、蛋白質陣列分析、蛋白質分泌法(例如藉由血漿凝固檢定或PDC條件培養基分析(例如藉由酶聯免疫吸附檢定法(Enzyme Linked ImmunoSorbent Assay,ELISA)))、混合淋巴球反應(例如作為PBMC刺激作用之量測)及/或其他該項技術領域中所習知的方法。 The precursor cells of the present invention, such as PPDC, can be characterized by: for example, growth characteristics (eg, population doubling ability, doubling time, number of passages to aging), karyotype analysis (eg, normal karyotype; maternal or newborn lineage), flow Cytometry (eg FACS analysis), immunohistochemistry and / or immunocytochemistry (eg detection of epitopes), gene expression profiles (eg gene chip array analysis; polymerase chain reaction (eg reverse transcriptase PCR, Real-time PCR and traditional PCR)), protein array analysis, protein secretion method (e.g. by plasma coagulation assay or PDC conditioned medium analysis (e.g. by Enzyme Linked ImmunoSorbent Assay (ELISA))), mixed lymphocytes Spherical response (e.g. as a measure of PBMC stimulation) and / or other methods known in the art.
衍生自臍組織之PPDC之實例係於2004年6月10日寄存於美國菌種保存中心(ATCC,10801 University Boulevard,Manassas,VA,20110),且所獲派之ATCC存取號如下:(1)細胞株代號UMB 022803(P7)所獲派之存取號為PTA-6067;以及(2)細胞株代號UMB 022803(P17)所獲派之存取號為PTA-6068。衍生自胎盤組織之PPDC之實例係寄存於美國菌種保存中心(ATCC,Manassas,Va.),且所獲派之ATCC存取號如下:(1)細胞株代號PLA 071003(P8)係於2004年6月15日寄存且所獲派之存取號為PTA-6074;(2)細胞株代號PLA 071003(P11)於2004年6月15日寄存且所獲派之存取號為PTA-6075;以及(3)細胞株代號PLA 071003(P16)於2004年6月16日寄存且所獲派之存取號為PTA-6079。 An example of PPDC derived from umbilical tissue was deposited with the American Type Culture Preservation Center (ATCC, 10801 University Boulevard, Manassas, VA, 20110) on June 10, 2004, and the assigned ATCC access number is as follows: (1 ) The cell line code UMB 022803 (P7) is assigned the access number PTA-6067; and (2) the cell line code UMB 022803 (P17) is assigned the access number PTA-6068. Examples of PPDCs derived from placental tissues are deposited with the American Type Culture Collection (ATCC, Manassas, Va.), And the ATCC access numbers assigned are as follows: (1) Cell line code PLA 071003 (P8) was in 2004 Deposited on June 15, 2014 and assigned access number PTA-6074; (2) Cell line code PLA 071003 (P11) was deposited on June 15, 2004 and assigned access number PTA-6075 ; And (3) The cell line code PLA 071003 (P16) was deposited on June 16, 2004 and the assigned access number was PTA-6079.
在各種實施例中,PPDC擁有以下生長特徵中之一或多者:(1)在培養中需要L-纈胺酸以供生長;(2)它們能夠生長於含有約5%至至少約20%之氧的氛圍中;(3)它們在培養中於達到衰老前具有至少約40次倍增之潛力;以及(4)它們在經塗布或未經塗布的組織培養容器上附著並擴增,其中經塗布之組織培養容器包含明 膠、層黏蛋白、膠原蛋白、聚鳥胺酸、玻璃連接蛋白、或纖連蛋白素之塗層。 In various embodiments, PPDC possesses one or more of the following growth characteristics: (1) L-valine is required for growth in culture; (2) They can grow to contain about 5% to at least about 20% In the atmosphere of oxygen; (3) they have the potential of at least about 40 doublings before reaching senescence in culture; and (4) they attach and expand on coated or uncoated tissue culture vessels, where The coated tissue culture container contains a coating of gelatin, laminin, collagen, polyguanylic acid, glass connexin, or fibronectin.
在某些實施例中,PPDC擁有正常核型,其在細胞繼代時維持。核型分析尤其可用於識別並區分衍生自胎盤之新生兒與母體細胞。核型分析方法為所屬技術領域中具有通常知識者可使用且習知者。 In certain embodiments, PPDC possesses a normal karyotype, which is maintained during cell passage. Karyotyping is particularly useful for identifying and distinguishing neonates and maternal cells derived from the placenta. The karyotype analysis method can be used and known by those with ordinary knowledge in the technical field.
在其他實施例中,PPDC可藉由生產某些蛋白質來表徵,包括:(1)生產波形蛋白(vimentin)與α-平滑肌肌動蛋白(alpha-smooth muscle actin)中之至少一者;以及(2)生產CD10、CD13、CD44、CD73、CD90、PDGFr-α、PD-L2與HLA-A、HLA-B、HLA-C細胞表面標記中之至少一者,如藉由流動式細胞測量術所偵測。在其他實施例中,PPDC可藉由不生產下列之至少一者來表徵:CD31、CD34、CD45、CD80、CD86、CD117、CD141、CD178、B7-H2、HLA-G以及HLA-DR、HLA-DP、HLA-DQ細胞表面標記,如藉由流動式細胞測量術所偵測。尤其較佳者為生產波形蛋白與α-平滑肌肌動蛋白的細胞。 In other embodiments, PPDC can be characterized by producing certain proteins, including: (1) producing at least one of vimentin and alpha-smooth muscle actin; and ( 2) Production of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr-α, PD-L2 and HLA-A, HLA-B, HLA-C cell surface markers, such as by flow cytometry Detect. In other embodiments, PPDC can be characterized by not producing at least one of the following: CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR, HLA- DP, HLA-DQ cell surface markers, as detected by flow cytometry. Particularly preferred are cells that produce vimentin and α-smooth muscle actin.
在其他實施例中,PPDC可藉由針對編碼下列之至少一者的基因之基因表現(相對於纖維母細胞、間葉幹細胞或髂骨崤骨髓細胞之人類細胞)係經增加來表徵:介白素8;內質網蛋白(reticulon)1;趨化激素(C--X--C模體)配體1(黑色素瘤生長刺激活性物,α);趨化激素(C--X--C模體)配體6(顆粒性細胞趨化蛋白質2);趨化激素(C--X--C模體)配體3;腫瘤壞死因子,α-誘發 蛋白質3;C型凝集素超家族成員2;威爾姆斯(Wilms)腫瘤1;醛去氫酶1家族成員A2;腎素(renin);氧化低密度脂蛋白受體1;智人(Homo sapiens)殖株IMAGE:4179671;蛋白質激酶C ζ;假想蛋白質DKFZp564F013;卵巢癌向下調控因子1;及殖株DKFZp547k1113之智人基因。在一實施例中,衍生自臍帶組織之PPDC可藉由針對編碼下列之至少一者的基因之基因表現(相對於纖維母細胞、間葉幹細胞或髂骨崤骨髓細胞之人類細胞)係經增加來表徵:介白素8;內質網蛋白(reticulon)1;或趨化激素(C--X--C模體)配體3。在另一實施例中,衍生自胎盤組織之PPDC可藉由針對編碼腎素或氧化低密度脂蛋白受體1之至少一者的基因之基因表現(相對於纖維母細胞、間葉幹細胞或髂骨崤骨髓細胞之人類細胞)係經增加來表徵。 In other embodiments, PPDC can be characterized by an increase in gene expression (relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells) encoding genes encoding at least one of: Retinol 8; Reticulon 1; Chemokine (C--X--C motif) ligand 1 (melanoma growth stimulating active substance, alpha); Chemokine (C--X-- C motif) ligand 6 (granulocyte chemoattractant protein 2); chemokine (C--X--C motif) ligand 3; tumor necrosis factor, alpha-induced protein 3; C-type lectin super Family member 2; Wilms tumor 1; aldehyde dehydrogenase 1 family member A2; renin; oxidized low-density lipoprotein receptor 1; Homo sapiens strain IMAGE: 4179671; Protein kinase C ζ; hypothetical protein DKFZp564F013; ovarian cancer down-regulator 1; and Homo sapiens gene of strain DKFZp547k1113. In one embodiment, PPDC derived from umbilical cord tissue can be increased by gene expression (relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells) encoding genes encoding at least one of the following To characterize: interleukin 8; reticulon 1 (reticulon) 1; or chemokine (C--X--C motif) ligand 3. In another embodiment, PPDC derived from placental tissue may be expressed by a gene targeting at least one gene encoding renin or oxidized low density lipoprotein receptor 1 (relative to fibroblasts, mesenchymal stem cells, or iliac Human cells of bone marrow cells are characterized by increase.
在其他實施例中,PPDC可藉由針對編碼下列之至少一者的基因之基因表現(相對於纖維母細胞、間葉幹細胞或髂骨崤骨髓細胞之人類細胞)係經減少來表徵:矮小同源盒2;熱休克27kDa蛋白質2;趨化激素(C--X--C模體)配體12(基質細胞衍生因子1);彈性蛋白(瓣上主動脈狹窄,威廉-波倫(Williams-Beuren)症候群);智人(Homo sapiens)mRNA;cDNA DKFZp586M2022(來自殖株DKFZp586M2022);間葉同源盒2(生長停滯特異性同源盒(growth arrest-specific homeo box));sine oculis同源盒同源物1(果蠅(Drosophila));αB水晶體蛋白;形態發生紊亂關聯活化物2(disheveled associated activator of morphogenesis 2);DKFZP586B2420蛋白質;類似於neuralin 1者;結合四素 (tetranectin,纖維蛋白溶酶原(plasminogen)結合蛋白質);src同源體3(SH3)與多半胱胺酸區(cysteine rich domain);膽固醇25-羥化酶;矮小相關轉錄因子3(runt-related transcription factor 3);介白素11受體,α;原膠原蛋白C-內肽酶增強子(procollagen C-endopeptidase enhancer);捲曲同源物7(frizzled homolog 7)(果蠅);假想基因BC008967;第三型膠原蛋白α1;固生蛋白C(tenascin C,hexabrachion);易洛魁族同源盒蛋白質5(iroquois homeobox protein 5);希菲斯特蛋白(hephaestin);整合素β8;突觸囊泡醣蛋白(synaptic vesicle glycoprotein)2;神經胚細胞瘤,致腫瘤性抑制因子1;類胰島素生長因子結合蛋白質2,36kDa;智人cDNA FLJ12280 fis,殖株MAMMA1001744;類細胞介素受體因子1;鉀中間物/小電導鈣活化通道,次家族N,成員4;整合素β7;具有PDZ-結合模體之轉錄共活化物(TAZ);sine oculis同源盒同源物2(果蠅);KIAAI034蛋白質;囊泡相關膜蛋白質5(肌短蛋白(myobrevin));含EGF類fibulin細胞外基質蛋白質(EGF-containing fibulin-like extracellular matrix protein)1;早期生長反應蛋白質(early growth response)3;無背端同源盒(distal-less homeo box)5;假想蛋白質FLJ20373;醛基-酮基還原酶家族1,成員C3(3-α羥類固醇去氫酶(3-alpha hydroxysteroid dehydrogenase),II型);雙聚醣(biglycan);具有PDZ-結合模體之轉錄共活化物(TAZ);纖連蛋白素1;腦啡肽前質(proenkephalin);整合素,類β1(具有類EGF重複區);智人mRNA全長插入物cDNA殖株EUROIMAGE 1968422;EphA3;KIAA0367 蛋白質;利尿鈉胜肽受體C(natriuretic peptide receptor C)/鳥苷酸環化酶C(guanylate cyclase C)(心房利尿鈉胜肽受體C(atrionatriuretic peptide receptor C));假想蛋白質FLJ14054;智人(Homo sapiens)mRNA;cDNA DKFZp564B222(來自殖株DKFZp564B222);BCL2/類腺病毒E1B 19kDa交互作用蛋白質3(adenovirus E1B 19kDa interacting protein 3-like);AE結合蛋白質1;以及細胞色素c氧化酶次單元VIIa多肽1(肌肉)。 In other embodiments, PPDC can be characterized by a reduction in gene expression (relative to human cells of fibroblasts, mesenchymal stem cells, or iliac bone marrow cells) that encodes at least one of the following genes: dwarf Source box 2; heat shock 27kDa protein 2; chemokine (C--X--C phantom) ligand 12 (stromal cell-derived factor 1); elastin (superior valve aortic stenosis, Williams-Polen (Williams) -Beuren) syndrome; Homo sapiens mRNA; cDNA DKFZp586M2022 (from the strain DKFZp586M2022); mesenchymal homeobox 2 (growth arrest-specific homeo box); sine oculis homeobox Homolog 1 (Drosophila); αB aquacris; morphogenetic disorder associated activator 2 (disheveled associated activator of morphogenesis 2); DKFZP586B2420 protein; similar to neuralin 1; bound tetranectin (tetranectin, fibrinolytic) Plasminogen binding protein); src homolog 3 (SH3) and cysteine rich domain; cholesterol 25-hydroxylase; runt-related transcription factor 3; Albumin 11 receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypothetical gene BC008967; type III collagen α1; Tenascin C (hexabrachion); Iroquois homeobox protein 5 (iroquois homeobox protein 5); Hephaestin (hephaestin); integrin β8; synaptic vesicle glycoprotein (synaptic vesicle glycoprotein) 2; neuroblastoma, tumorigenic inhibitory factor 1; insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNA FLJ12280 fis, strain MAMMA1001744; cytokinin receptor factor 1; potassium intermediate / Small conductivity calcium-activated channel, subfamily N, member 4; integrin β7; transcription coactivator (TAZ) with PDZ-binding motif; sine oculis homeobox homolog 2 (Drosophila); KIAAI034 protein; capsule Follicle-associated membrane protein 5 (myobrevin); EGF-containing fibulin-like extracellular matrix protein 1; early growth response protein 3; no dorsal end Source box (distal-less homeo box) 5; hypothetical protein FLJ20373; aldehyde-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II); disaccharide ( biglycan); transcription coactivator (TAZ) with PDZ-binding motif; fibronectin 1; proenkephalin; integrin, β1 like (with EGF-like repeat); Homo sapiens mRNA full length Insert cDNA strain EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptor C (guantriate cyclase C) (guanylate cyclase C) (atrionatriuretic peptide C (atrionatriuretic peptide receptor C)); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222 (from strain DKFZp564B222); BCL2 / adenovirus E1B 19kDa interacting protein 3 (adenovirus E1B 19kDa interacting protein 3-like); AE Binding protein 1; and cytochrome c oxidase subunit VIIa polypeptide 1 (muscle).
在其他實施例中,PPDC可藉由分泌選自MFG-E8、Gas6、TSP-1及TSP-2之橋分子來表徵。此外,衍生自臍帶組織之PPDC可藉由分泌MCP-1、IL-6、IL-8、GCP-2、HGF、KGF、FGF、HB-EGF、BDNF、TPO、MIP1b、I309、RANTES、MDC、及TIMP1中之至少一者來表徵。在一些實施例中,衍生自臍帶組織之PPDC可藉由不分泌TGF-β2、ANG2、PDGFbb、MIP1a及VEGF中之至少一者來表徵,如藉由ELISA所偵測。在替代實施例中,衍生自胎盤組織之PPDC可藉由分泌MCP-1、IL-6、IL-8、GCP-2、HGF、KGF、HB-EGF、BDNF、TPO、MIP1a、RANTES、及TIMP1中之至少一者,且不分泌TGF-β2、MIP1b、ANG2、PDGFbb、FGF、及VEGF中之至少一者來表徵,如藉由ELISA所偵測。在進一步實施例中,PPDC不表現hTERT或端粒酶。 In other embodiments, PPDC can be characterized by secreting a bridge molecule selected from MFG-E8, Gas6, TSP-1, and TSP-2. In addition, PPDC derived from umbilical cord tissue can be secreted by MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, I309, RANTES, MDC, And at least one of TIMP1. In some embodiments, PPDC derived from umbilical cord tissue can be characterized by not secreting at least one of TGF-β2, ANG2, PDGFbb, MIP1a, and VEGF, as detected by ELISA. In alternative embodiments, PPDC derived from placental tissue can be secreted by MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1 At least one of them is not characterized by secretion of at least one of TGF-β2, MIP1b, ANG2, PDGFbb, FGF, and VEGF, as detected by ELISA. In a further embodiment, PPDC does not express hTERT or telomerase.
在較佳實施例中,細胞包含二或更多種以上列舉之生長、蛋白質/表面標記生產、基因表現或物質分泌之特徵。更佳者為該些包含三、四、或五或更多種該等特徵之細胞。再佳者為包含六、 七或八或更多種該等特徵之PPDC。再佳者為該些包含所有上述特徵之細胞。 In a preferred embodiment, the cell contains two or more of the characteristics listed above for growth, protein / surface marker production, gene expression, or substance secretion. More preferably, the cells contain three, four, or five or more of these characteristics. Even better is a PPDC containing six, seven, or eight or more of these characteristics. Even better are those cells that contain all the above characteristics.
在特別較佳的實施例中,細胞係分離自實質上不含血液之人類臍帶組織,該等細胞能夠在培養物中擴增、不生產CD117或CD45、且不表現hTERT或端粒酶。在一實施例中,細胞不生產CD117及CD45且可選地亦不表現hTERT及端粒酶。在另一實施例中,細胞不表現hTERT及端粒酶。在另一實施例中,細胞係分離自實質上不含血液之人類臍帶組織、能夠在培養物中擴增、不生產CD117或CD45、且不表現hTERT或端粒酶,且具有以下特徵之一或多者:表現CD10、CD13、CD44、CD73、及CD90;不表現CD31或CD34;相對於人類纖維母細胞、間葉幹細胞或髂骨崤骨髓細胞,表現增加水準之介白素8或內質網蛋白1;且具有分化之潛能。 In a particularly preferred embodiment, the cell line is isolated from human umbilical cord tissue that is substantially free of blood. These cells can be expanded in culture, do not produce CD117 or CD45, and do not express hTERT or telomerase. In one embodiment, the cells do not produce CD117 and CD45 and optionally also do not express hTERT and telomerase. In another embodiment, the cells do not express hTERT and telomerase. In another embodiment, the cell line is isolated from human umbilical cord tissue that is substantially free of blood, can be expanded in culture, does not produce CD117 or CD45, and does not express hTERT or telomerase, and has one of the following characteristics Or more: express CD10, CD13, CD44, CD73, and CD90; do not express CD31 or CD34; relative to human fibroblasts, mesenchymal stem cells, or iliac bone marrow cells, exhibit increased levels of interleukin 8 or endoplasmic Reticulin 1; and has the potential to differentiate.
在本發明之數個態樣中所使用之目前較佳的細胞為具有上述特徵之產後細胞,並且尤其是其中該些細胞具有正常核型並且在繼代時維持正常核型者,而進一步是其中該些細胞表現下列各標記者:CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C,其中該些細胞生產免疫學上可偵測且對應上列標記之蛋白質。再佳者為除了前述者外不生產對應於任何下列標記之蛋白質之該些細胞:CD31、CD34、CD45、CD117、CD141或HLA-DR、HLA-DP、HLA-DQ如由流動式細胞測量術所偵測。在進一步較佳實施例中,該些細胞不表現hTERT或端粒酶。 The currently preferred cells used in several aspects of the present invention are post-natal cells with the above characteristics, and especially those in which these cells have a normal karyotype and maintain the normal karyotype at subsequent passages, and further The cells showed the following markers: CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA-B, HLA-C, and the production of these cells was immunologically detectable and corresponding to List of labeled proteins. Even better are those cells that do not produce proteins corresponding to any of the following markers except for the aforementioned: CD31, CD34, CD45, CD117, CD141 or HLA-DR, HLA-DP, HLA-DQ as determined by flow cytometry Detected. In a further preferred embodiment, these cells do not express hTERT or telomerase.
具有譜系分化的潛能而導致各種表型之某些細胞係不穩定且因此可自行分化。目前較佳用於本發明的細胞為不會例如沿著神經譜系自行分化之細胞。當較佳細胞生長於生長培養基中時,生產在其表面上之細胞標記,及其各種基因之表現模式(如使用Affymetrix GENECHIP所測定)為實質上穩定的。經由多次族群倍增的繼代,該些細胞例如在其表面標記特徵方面仍保持實質上恆定。 Certain cell lines with various phenotypes have the potential for lineage differentiation and are therefore unstable and therefore can differentiate on their own. The cells currently preferred for use in the present invention are cells that do not differentiate themselves, for example, along the neural lineage. When the preferred cells are grown in growth medium, the cell markers produced on their surface, and the expression patterns of their various genes (as determined using Affymetrix GENECHIP) are substantially stable. Through multiple population doublings, these cells remain substantially constant, for example, in terms of their surface marking characteristics.
然而,PPDC之一個特徵在於可藉由使其經歷分化誘導細胞培養條件來有意地誘導其分化成各種譜系表型。在治療某些眼變性病狀的用途中,可使用所屬技術領域中已知的一或多種方法誘導PPDC分化成神經表型。舉例而言,如本文所例示,可將PPDC接種於經層黏蛋白塗布之培養瓶中之含有B27(B27補充物,Invitrogen)、L-麩醯胺酸及青黴素/鏈黴素之Neurobasal-A培養基(Invitrogen,Carlsbad,Calif.),其組合在本文稱為神經前驅擴增(NPE)培養基。NPE培養基可進一步補充有bFGF及/或EGF。或者,可藉由以下誘導PPDC體外分化:(1)將PPDC與神經前驅細胞共培養;或(2)使PPDC於神經前驅細胞條件培養基中生長。 However, a characteristic of PPDC is that it can be intentionally induced to differentiate into various lineage phenotypes by subjecting it to differentiation-inducing cell culture conditions. In the treatment of certain ocular degenerative conditions, one or more methods known in the art can be used to induce the differentiation of PPDC into a neurophenotype. For example, as exemplified herein, PPDC can be inoculated into Neurobasal-A containing B27 (B27 supplement, Invitrogen), L-glutamic acid, and penicillin / streptomycin in a culture bottle coated with laminin Medium (Invitrogen, Carlsbad, Calif.), The combination of which is referred to herein as neural precursor expansion (NPE) medium. The NPE medium can be further supplemented with bFGF and / or EGF. Alternatively, in vitro differentiation of PPDC can be induced by: (1) co-culturing PPDC with neural precursor cells; or (2) growing PPDC in conditioned medium of neural precursor cells.
PPDC分化成神經表型可藉由具有伸長突起之雙極細胞形態來證明。經誘導之細胞群可針對巢蛋白(nestin)之存在染色為陽性。經分化之PPDC可藉由偵測巢蛋白、TuJ1(BIII微管蛋白)、GFAP、酪胺酸羥化酶、GABA、04及/或MBP來評估。在一些實施 例中,PPDC展現出形成神經球(neurosphere)之神經元幹細胞形成之三維體特徵的能力。 The differentiation of PPDC into a neurophenotype can be demonstrated by the morphology of bipolar cells with elongated processes. The induced cell population can be stained positive for the presence of nestin. Differentiated PPDC can be evaluated by detecting nestin, TuJ1 (BIII tubulin), GFAP, tyrosine hydroxylase, GABA, 04 and / or MBP. In some embodiments, PPDC exhibits the ability to form the three-dimensional features of neurosphere stem cells that form neurospheres.
本發明之另一態樣之特徵在於前驅細胞諸如產後衍生細胞之族群。產後衍生細胞可分離自胎盤或臍組織。在較佳實施例中,細胞群包含上文所述之PPDC,且這些細胞群係描述於下節中。 Another aspect of the invention is characterized by a population of precursor cells such as postpartum derived cells. Postpartum-derived cells can be isolated from placenta or umbilical tissue. In a preferred embodiment, the cell populations include PPDC as described above, and these cell population lines are described in the next section.
在一些實施例中,細胞群為異質性。本發明之異質性細胞群可包含至少約5%、10%、20%、30%、40%、50%、60%、70%、80%、90%或95%的細胞。本發明之異質性細胞群可進一步包含前驅細胞(產後衍生細胞)、或其他前驅細胞,諸如上皮或神經前驅細胞,或其可進一步包含經完全分化之細胞。 In some embodiments, the cell population is heterogeneous. The heterogeneous cell population of the present invention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% cells. The heterogeneous cell population of the present invention may further include precursor cells (postpartum-derived cells), or other precursor cells, such as epithelial or neural precursor cells, or it may further include fully differentiated cells.
在一些實施例中,該族群實質上為均質性,即實質上僅包含PPDC(較佳為至少約96%、97%、98%、99%或更多的細胞)。在一些實施例中,細胞群係均質性。在實施例中,本發明之均質性細胞群可包含臍衍生細胞或胎盤衍生細胞。臍衍生細胞之均質性族群較佳為不含母體譜系細胞。胎盤衍生細胞之均質性族群可為新生兒或母體譜系。細胞群之均質性可藉由任何該項技術領域中所習知的方法來達成,例如根據習知方法藉由細胞分選(例如流動式細胞測量術)或藉由殖株擴增達成。因此,較佳均質性PPDC族群可包 含產後衍生細胞之殖株細胞系。當分離出具有高度所欲功能性之細胞殖株時,此類族群為尤其有用的。 In some embodiments, the ethnic group is substantially homogenous, that is, it substantially contains only PPDC (preferably at least about 96%, 97%, 98%, 99% or more cells). In some embodiments, the cell population line is homogeneous. In embodiments, the homogeneous cell population of the present invention may comprise umbilical derived cells or placental derived cells. The homogeneous population of umbilical derived cells is preferably free of maternal lineage cells. The homogeneous population of placental-derived cells can be neonatal or maternal lineage. The homogeneity of the cell population can be achieved by any method known in the art, for example, by cell sorting (eg, flow cytometry) or by plant expansion according to conventional methods. Therefore, it is preferred that the homogeneous PPDC population can contain a colony cell line of postpartum-derived cells. Such ethnic groups are particularly useful when isolating cell strains with highly desired functionality.
本文亦提供在一或多種因子存在下,或在刺激幹細胞沿著所欲路徑(例如,神經、上皮)分化之條件下培養之細胞群。此類因子為所屬技術領域中已知的且熟悉該項技藝人士將會瞭解到,判定合適的分化條件可以常規實驗完成。此類條件之最佳化可藉由統計實驗設計及分析來完成,例如回應面方法論(response surface methodology)允許同時最佳化例如在生物培養物中的多個變數。目前較佳因子包括但不限於以下因子,諸如生長或營養因子、去甲基化劑、與神經或上皮譜系細胞共培養或於神經或上皮譜系細胞條件培養基中培養,以及所屬技術領域中已知用以刺激幹細胞沿著這些路徑分化的其他條件(關於可用於神經分化之因子,參見例如Lang,K.J.D.等人,2004,J.Neurosci.Res.76:184-192;Johe,K.K.等人,1996,Genes Devel.10:3129-3140;Gottleib,D.,2002,Ann.Rev.Neurosci.25:381-407)。 Also provided herein are cell populations cultured in the presence of one or more factors, or under conditions that stimulate the differentiation of stem cells along a desired path (eg, nerve, epithelium). Such factors are known in the art and those skilled in the art will understand that the determination of appropriate differentiation conditions can be accomplished through routine experiments. The optimization of such conditions can be accomplished by statistical experiment design and analysis, such as response surface methodology allowing simultaneous optimization of multiple variables, such as in biological cultures. Currently preferred factors include, but are not limited to, factors such as growth or trophic factors, demethylating agents, co-cultivation with nerve or epithelial lineage cells, or culture in nerve or epithelial lineage cell conditioned medium, and are known in the art Other conditions used to stimulate stem cells to differentiate along these pathways (for factors that can be used for neural differentiation, see, for example, Lang, KJD et al., 2004, J. Neurosci. Res. 76: 184-192; Johe, KK et al., 1996 , Genes Devel. 10: 3129-3140; Gottleib, D., 2002, Ann. Rev. Neurosci. 25: 381-407).
在一個態樣中,本發明提供來自經培養之前驅細胞諸如產後衍生細胞、或其他前驅細胞之條件培養基,其如下文所述用於體外及體內。此類條件培養基之使用允許由細胞分泌之有益營養因子同種異體地用於患者中,而無需引入可能引發排斥、或其他不良免疫反應之完整細胞。條件培養基係藉由於培養基中培養細胞(諸 如細胞群),隨後自培養基移除該些細胞來製備。在某些實施例中,產後細胞為UTC或PDC,更佳為hUTC。 In one aspect, the invention provides a conditioned medium from cultured precursor cells such as postpartum-derived cells, or other precursor cells, which is used in vitro and in vivo as described below. The use of such conditioned medium allows the beneficial nutrient factors secreted by the cells to be used allogeneically in patients without introducing whole cells that may trigger rejection or other adverse immune reactions. The conditioned medium is prepared by culturing cells (such as cell populations) in the medium, and then removing the cells from the medium. In some embodiments, the postpartum cells are UTC or PDC, more preferably hUTC.
由上文所述之細胞群所製備之條件培養基可按原樣使用、經進一步濃縮例如藉由超濾或凍乾、或甚至乾燥後使用、經部分純化後使用、如所述技術領域中已知與醫藥上可接受之載劑或稀釋劑組合使用、或與其他化合物諸如生物製品例如醫藥上可用的蛋白質組成物組合使用。條件培養基可在體外或體內單獨使用,或例如與自體或同基因活細胞一起使用。條件培養基若經引入體內,可被引入局部治療位點、或引入遠端以提供例如需要的細胞生長或營養因子給患者。 The conditioned medium prepared from the cell population described above can be used as is, further concentrated, for example by ultrafiltration or lyophilization, or even dried, used after partial purification, as known in the technical field Used in combination with a pharmaceutically acceptable carrier or diluent, or in combination with other compounds such as biological products such as pharmaceutically usable protein compositions. The conditioned medium can be used alone in vitro or in vivo, or for example with autologous or syngeneic living cells. If the conditioned medium is introduced into the body, it can be introduced into a local treatment site or distally to provide, for example, the desired cell growth or nutritional factors to the patient.
先前已證明,人類臍帶組織衍生細胞改良視覺功能且改善視網膜變性(參見US 2010/0272803)。亦已證明,產後衍生細胞可在RCS模型中用於促進光受體救援且因此保留光受體。(參見US 2010/0272803)。將hUTC視網膜下注射至RCS大鼠眼睛中改善了視敏度並緩解了視網膜變性。 It has previously been shown that human umbilical cord tissue-derived cells improve visual function and improve retinal degeneration (see US 2010/0272803). It has also been shown that postpartum-derived cells can be used in the RCS model to promote photoreceptor rescue and thus retain photoreceptors. (See US 2010/0272803). Subretinal injection of hUTC into the eyes of RCS rats improved visual acuity and relieved retinal degeneration.
如本文所提供,製備hUTC條件培養基之各種製劑且評估其吞噬救援活性。發現接種密度及培養條件影響條件培養基之活性水準。以hUTC於血清中(CM1)而言,將hUTC以5,000個存活細胞/cm2接種於T75細胞培養瓶中之hUTC生長培養基(DMEM低葡萄糖+15% FBS+4mM L-麩醯胺酸)中,且培養24小時。將培養基更換成21mL DMEM/F12完全培養基(DMEM:F12培養基+10% FBS+Pen(50U/ml)/Strep(50μg/ml)),再將細胞培養54小時,且收集培養物上清液並在-70℃下冷凍(凍存)。 As provided herein, various formulations of hUTC conditioned medium were prepared and evaluated for phagocytosis rescue activity. It was found that inoculation density and culture conditions affect the activity level of conditioned medium. In terms of hUTC in serum (CM1), inoculate hUTC at 5,000 viable cells / cm 2 in hUTC growth medium (DMEM low glucose + 15% FBS + 4mM L-glutamic acid) in T75 cell culture flasks And cultivate for 24 hours. The medium was replaced with 21 mL of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)), the cells were cultured for 54 hours, and the culture supernatant was collected and Freeze (freeze) at -70 ° C.
以無血清培養基(CM1無血清)而言,在第2天將培養基以21mL DMEM/F12無血清培養基(DMEM:F12培養基+Pen(50U/ml)/Strep(50μg/ml))添補。具有或不具有血清之CM1使吞噬活性恢復(圖1A至圖1B及圖2)。以製備具有血清之CM1之相同程序製備另一條件培養基(CM2),除了細胞係培養於每瓶具有63mL培養基之T225瓶,且在更換培養基之後培養時間為48小時。然而,此培養基不具活性。(圖3A)。以和CM2相同的條件製備CM3,但使用10,000個存活細胞/cm2,且CM3刺激失養性RPE中之吞噬。(圖3B至圖3D)。 As for the serum-free medium (CM1 serum-free), the medium was supplemented with 21 mL of DMEM / F12 serum-free medium (DMEM: F12 medium + Pen (50 U / ml) / Strep (50 μg / ml)) on the second day. CM1 with or without serum restored phagocytic activity (Figure 1A to Figure 1B and Figure 2). Another conditioned medium (CM2) was prepared in the same procedure as CM1 with serum, except that the cell lines were cultured in T225 bottles with 63 mL of medium per bottle, and the culture time was 48 hours after changing the medium. However, this medium is not active. (Figure 3A). CM3 was prepared under the same conditions as CM2, but 10,000 viable cells / cm 2 were used , and CM3 stimulated phagocytosis in the deferent RPE. (Figures 3B to 3D).
在所測試之條件中,發現CM2缺乏活性(圖3A)。相較於CM1有54小時的培養時間,CM2的培養時間縮短為48h。相較於CM2,CM3係藉由將細胞接種密度加倍並維持更換培養基後之相同培養時間來製備,且發現CM3具有活性。因此,為獲得活性CM,初始細胞接種密度及更換培養基後之細胞培養時間為兩個重要條件。 In the tested conditions, CM2 was found to lack activity (Figure 3A). Compared with CM1, which has a cultivation time of 54 hours, the cultivation time of CM2 is shortened to 48 hours. Compared to CM2, CM3 was prepared by doubling the cell seeding density and maintaining the same incubation time after changing the medium, and CM3 was found to be active. Therefore, in order to obtain active CM, the initial cell seeding density and the cell culture time after medium replacement are two important conditions.
來自皇家外科學院(RCS)大鼠之視網膜色素上皮(RPE)細胞由於Mertk基因之突變而具有視桿細胞外節(ROS)之缺陷性吞噬。Mertk為受體酪胺酸激酶(RTK)家族之成員且被認為在RPE吞噬上有其角色。鹼性纖維母細胞生長因子(bFGF)為FGF RTK之配體,經顯示可誘導來自RCS大鼠之經培養RPE細胞中之吞噬能力 (McLaren等人,FEBS Letters,1997;412:21-29)。在本發明之一實施例中,hUTC救援失養性RPE之吞噬係透過RTK配體之分泌、活化RTK傳訊、及增強其他吞噬相關受體之傳訊。 Retinal pigment epithelium (RPE) cells from the Royal College of Surgeons (RCS) rats have defective phagocytosis of the rod outer segment (ROS) due to mutations in Mertk gene. Mertk is a member of the receptor tyrosine kinase (RTK) family and is thought to have its role in RPE phagocytosis. Basic fibroblast growth factor (bFGF) is a ligand for FGF RTK and has been shown to induce phagocytic ability in cultured RPE cells from RCS rats (McLaren et al., FEBS Letters , 1997; 412: 21-29) . In one embodiment of the present invention, the phagocytosis of hUTC to rescue defertile RPE is through the secretion of RTK ligands, activation of RTK signaling, and enhancement of signaling by other phagocytic related receptors.
在本發明之一實施例中,RTK配體BDNF、HB-EGF、PDGF-DD、蝶素(ephrin)A4、HGF、及蝶素B2對於RCS失養性RPE細胞吞噬具有救援作用。在一具體實施例中,BDNF、PDGF-DD、及蝶素B2具有陽性救援作用。(參見圖5A、6A至6B及7A至7B)。 In one embodiment of the present invention, the RTK ligands BDNF, HB-EGF, PDGF-DD, ephrin A4, HGF, and Pterin B2 have a rescue effect on phagocytosis of RCS-dystrophic RPE cells. In a specific embodiment, BDNF, PDGF-DD, and Pterin B2 have a positive rescue effect. (See FIGS. 5A, 6A to 6B and 7A to 7B).
非RTK配體活化與RTK不同之受體,且對吞噬不具有和RTK配體類似的作用(圖8A至8C及圖9)。hUTC已顯示出會分泌玻璃連接蛋白、內皮素-1、TGF-β1、及IL-6。玻璃連接蛋白之受體包括αvβ3及αvβ5整合素。Finnemann等人報告,RPE細胞對ROS之吞噬需要αvβ5整合素(Finnemann等人,1997,見前文)。儘管hUTC CM提高失養性RPE細胞之吞噬,但是內皮素-1、TGF-β1或IL-6(濃度200ng/mL)、及玻璃連接蛋白(各種濃度)對RCS RPE吞噬無作用(圖8A至8C,圖9)。 The non-RTK ligand activates a receptor different from RTK and does not have a similar effect on phagocytosis as the RTK ligand (Figures 8A to 8C and Figure 9). hUTC has been shown to secrete vitronectin, endothelin-1, TGF-β1, and IL-6. Receptors for vitronectin include αvβ3 and αvβ5 integrin. Finnemann et al. Reported that RPE cells require αvβ5 integrin for phagocytosis of ROS (Finnemann et al., 1997, see above). Although hUTC CM increased the phagocytosis of dystrophic RPE cells, endothelin-1, TGF-β1 or IL-6 (concentration 200 ng / mL), and vitronectin (various concentrations) had no effect on RCS RPE phagocytosis (Figures 8A to 8C , Figure 9).
利用經條件培養基處理及未處理之失養性RPE的RNA分析所進行的基因表現概況顯示,hUTC表現15個RTK次家族內之多個RTK配體基因(圖10及表1-1)。hUTC亦表現橋分子基因,包括MFG-E8、Gas6、蛋白質S、TSP-1、及TSP-2(表1-3)。RCS RPE表現18個RTK次家族中之基因(表1-2)。在18個RTK次家族中有15個次家族對應hUTC中所表現之RTK配體基因。RCS RPE亦表現用於橋分子結合之受體基因,包括整合素αvβ3、αvβ5、Axl、Tyro3、MerTK、及CD36(表1-4)。 Gene expression profiles performed using RNA analysis of conditioned medium-treated and untreated dystrophic RPE showed that hUTC expressed multiple RTK ligand genes within 15 RTK subfamilies (Figure 10 and Table 1-1). hUTC also expresses bridge molecule genes, including MFG-E8, Gas6, protein S, TSP-1, and TSP-2 (Table 1-3). RCS RPE expresses genes in 18 RTK subfamilies (Table 1-2). Among the 18 RTK subfamilies, 15 subfamilies correspond to the RTK ligand genes expressed in hUTC. RCS RPE also expresses receptor genes for bridge molecule binding, including integrin αvβ3, αvβ5, Axl, Tyro3, MerTK, and CD36 (Table 1-4).
使用RNA-Seq接著進行資訊學數據分析之RCS RPE細胞與hUTC兩者之轉錄體概況顯示,RCS RPE細胞表現多個RTK基因,而hUTC表現多個RTK配體之基因(表1-1至1-4及表2-1)。特定言之,七個RTK次家族之RTK配體具有相對高的基因表現水準。此等配體包括BDNF(腦衍生神經營養因子)及NT3(神經營養蛋白3)-Trk家族之配體、HGF(肝細胞生長因子)-Met家族之配體、PDGF-DD(血小板衍生生長因子D型)及PDGF-CC(血小板衍生生長因子C型)-PDGF家族之配體、蝶素B2-Eph家族之配體、HB-EGF(肝素結合表皮生長因子)-ErbB家族之配體、GDNF(膠細胞衍生神經營養因子)-Ret家族之配體、以及聚集蛋白(agrin)-Musk家族之配體。 The transcript profile of both RCS RPE cells and hUTC using RNA-Seq followed by informatics data analysis showed that RCS RPE cells expressed multiple RTK genes, while hUTC expressed multiple RTK ligand genes (Tables 1-1 to 1 -4 and Table 2-1). In particular, the RTK ligands of the seven RTK subfamilies have a relatively high level of gene expression. These ligands include BDNF (brain-derived neurotrophic factor) and NT3 (neurotrophin 3)-ligands of the Trk family, HGF (hepatocyte growth factor)-ligands of the Met family, PDGF-DD (platelet-derived growth factor Type D) and PDGF-CC (platelet-derived growth factor type C) -ligands of PDGF family, ligands of Pterin B2-Eph family, HB-EGF (heparin-binding epidermal growth factor) -ligands of ErbB family, GDNF (Glial cell-derived neurotrophic factor)-ligand of the Ret family, and ligand of the agrin-Musk family.
在本發明之一實施例中,BDNF、NT3、HGF、及GDNF係經分泌於hUTC條件培養基中,且其水準相較於來自正常人類皮膚纖維母細胞(NHDF)及ARPE-19細胞之水準為高,如在ELISA檢定中所量測。(圖11A至11C及11F)。在另一實施例中,相較於NHDF或ARPE-19,hUTC分泌低水準之PDGF-CC及PDGF-DD(圖11D、11E)。在進一步實施例中,蝶素B2及HB-EGF未於hUTC、NHDF、及ARPE-19之條件培養基中偵測出,如在ELISA檢定中所量測。這些細胞不分泌這兩種蛋白質,或分泌水準低於ELISA檢定之偵測界限。hUTC、NHDF、及ARPE-19條件培養基中之聚集蛋白水準類 似於對照培養基;在所有條件培養基樣本中偵測出的聚集蛋白可能來自該培養基。 In one embodiment of the present invention, BDNF, NT3, HGF, and GDNF are secreted in conditioned medium of hUTC, and their levels are compared with those from normal human skin fibroblasts (NHDF) and ARPE-19 cells. High, as measured in the ELISA test. (Figures 11A to 11C and 11F). In another embodiment, hUTC secretes low levels of PDGF-CC and PDGF-DD compared to NHDF or ARPE-19 (Figures 11D, 11E). In a further embodiment, Pterin B2 and HB-EGF were not detected in the conditioned medium of hUTC, NHDF, and ARPE-19, as measured in the ELISA test. These cells do not secrete these two proteins, or the secretion level is below the detection limit of the ELISA test. The levels of agrin in hUTC, NHDF, and ARPE-19 conditioned medium are similar to the control medium; the agrin detected in all conditioned medium samples may come from this medium.
基於RCS RPE細胞之基於RNA-Seq的轉錄體概況分析,經分泌於hUTC條件培養基中之橋分子及其他因子之水準進一步證明對吞噬之作用,及因此對細胞凋亡之作用。如所示,RCS RPE細胞表現迄今已識別之辨識細胞凋亡細胞上「吃我」信號之許多受體之基因。此等受體包括清道夫受體(SR-A、LOX-1、CD68、CD36、CD14)、整合素(αvβ3及αvβ5)、Axl及Tyro3之受體酪胺酸激酶、LRP-1/CD91、及PS受體穩定素1(表3-1;改編自Erwig L-P and Henson PM,Cell Death and Differentiation 2008;15:243-250)。此外,hUTC表現許多橋分子基因,包括TSP-1、TSP-2、界面活性蛋白質D(SP-D)、MFG-E8、Gas6、載脂蛋白H(apolipoprotein H)、及膜聯蛋白1(annexin 1)。在本發明之一實施例中,hUTC於hUTC條件培養基中分泌MFG-E8、Gas6、TSP-1、TSP-2。(圖12A至12E及表3-2)。在另一實施例中,hUTC不分泌載脂蛋白H、SP-D、或膜聯蛋白I(表3-2)。在某些實施例中,hUTC以顯著高於NHDF及ARPE-19之水準分泌MFG-E8及TSP-2。(圖12A及12D)。 The RNA-Seq-based transcript profile analysis of RCS RPE cells based on the level of bridge molecules and other factors secreted in the hUTC conditioned medium further proved the effect on phagocytosis, and therefore on apoptosis. As shown, RCS RPE cells express the genes of many receptors that have been identified so far to recognize the "eat me" signal on apoptotic cells. These receptors include scavenger receptors (SR-A, LOX-1, CD68, CD36, CD14), integrin (αvβ3 and αvβ5), Axl and Tyro3 receptor tyrosine kinase, LRP-1 / CD91, And PS receptor stabilizer 1 (Table 3-1; adapted from Erwig LP and Henson PM, Cell Death and Differentiation 2008; 15: 243-250). In addition, hUTC expresses many bridge molecule genes, including TSP-1, TSP-2, interface active protein D (SP-D), MFG-E8, Gas6, apolipoprotein H (apolipoprotein H), and annexin 1 (annexin 1). In one embodiment of the present invention, hUTC secretes MFG-E8, Gas6, TSP-1, TSP-2 in hUTC conditioned medium. (Figures 12A to 12E and Table 3-2). In another embodiment, hUTC does not secrete apolipoprotein H, SP-D, or annexin I (Table 3-2). In certain embodiments, hUTC secretes MFG-E8 and TSP-2 at levels significantly higher than NHDF and ARPE-19. (Figures 12A and 12D).
在本發明之一態樣中,當對RCS RPE細胞饋以經hUTC CM預培養之ROS時,hUTC條件培養基刺激對ROS之吞噬。失養性RPE細胞之吞噬係經完全救援。如圖13A至13D中所示,未處理之失養性RPE細胞相較於正常RPE細胞具有減少之吞噬。在本發明之一實施例中,以hUTC CM預培養失養性RPE細胞救援 吞噬。即使在檢定期間不存在hUTC CM之情況下,此亦會發生。在本發明之實施例中,吞噬相關受體及其傳訊路徑在預培養期間係經上調。當hUTC CM存在於吞噬檢定期間時,不管失養性RPE細胞是否經hUTC CM預處理,皆觀察到穩固增強之吞噬。在一實施例中,請以經hUTC CM預處理之ROS之失養性RPE細胞恢復或救援吞噬。即使在吞噬檢定期間不存在hUTC CM之情況下,此亦會發生。在本發明之具體實施例中,hUTC CM可準備(prime)或改質ROS,其經由例如有利地促進吞噬之橋分子/調理素來增強ROS結合及內化。 In one aspect of the invention, when RCS RPE cells are fed with ROS pre-cultured with hUTC CM, hUTC conditioned medium stimulates phagocytosis of ROS. The phagocytosis of dystrophic RPE cells is completely rescued. As shown in Figures 13A to 13D, untreated defertile RPE cells have reduced phagocytosis compared to normal RPE cells. In one embodiment of the present invention, the dystrophic RPE cells are pre-cultured with hUTC CM to rescue phagocytosis. This will happen even if there is no hUTC CM during the verification period. In an embodiment of the present invention, phagocytosis-related receptors and their signaling pathways are up-regulated during pre-culture. When hUTC CM was present during the phagocytosis assay, steadily enhanced phagocytosis was observed regardless of whether the dystrophic RPE cells were pretreated with hUTC CM. In one embodiment, please restore or rescue phagocytosis with trophoblastic RPE cells pre-treated with hUTC CM. This will happen even if there is no hUTC CM during the phagocytosis test. In specific embodiments of the invention, hUTC CM can prime or modify ROS, which enhances ROS binding and internalization via, for example, bridge molecules / opsonins that advantageously promote phagocytosis.
在本發明之實施例中,橋分子MFG-E8、Gas6、TSP-1及TSP-2媒介RCS RPE細胞吞噬ROS。將失養性RPE細胞饋以經各種濃度之MFG-E8、Gas6、TSP-1或TSP-2預培養之ROS並檢定其吞噬,顯示救援對ROS之吞噬(圖14A至圖14H)。在具體實施例中,hUTC條件培養基經由分泌例如MFG-E8、Gas6、TSP-1及TSP-2之橋分子媒介RCS RPE吞噬救援。 In an embodiment of the present invention, the bridge molecules MFG-E8, Gas6, TSP-1 and TSP-2 mediate RCS RPE cells to phagocytose ROS. The dystrophic RPE cells were fed with ROS pre-cultured with various concentrations of MFG-E8, Gas6, TSP-1 or TSP-2 and tested for phagocytosis, showing rescue phagocytosis of ROS (Figure 14A to 14H). In a specific embodiment, the hUTC conditioned medium is phagocytosed and rescued by RCS RPE that secretes bridge molecules such as MFG-E8, Gas6, TSP-1 and TSP-2.
在一實施例中,RTK配體諸如BDNF、HGF及GDNF刺激RCS RPE中之hUTC媒介之吞噬救援。RTK配體BDNF、HGF及GDNF救援RCS RPE中之吞噬(圖14I至J)。重組RTK配體及橋分子蛋白可模擬hUTC CM之作用且恢復RCS RPE吞噬,且涉及RCS RPE中hUTC媒介之吞噬救援。 In one embodiment, RTK ligands such as BDNF, HGF, and GDNF stimulate hUTC-mediated phagocytosis rescue in RCS RPE. RTK ligands BDNF, HGF and GDNF rescue phagocytosis in RCS RPE (Figures 14I to J). Recombinant RTK ligands and bridge molecule proteins can mimic the role of hUTC CM and restore RCS RPE phagocytosis, and involve hUTC-mediated phagocytosis rescue in RCS RPE.
siRNA媒介基因靜默證明,hUTC中之BDNF、HGF、GDNF、MFG-E8、Gas6、TSP-1及TSP-2係經剔除(靜默)。模擬 或淩亂(scrambled)siRNA轉染對此等因子的hUTC分泌無作用。靶向MFG-E8、TSP-1、TSP-2、及HGF之siRNA產生幾乎100%剔除效率;BDNF及GDNF分別觀察到80%及65%剔除(圖15A至圖15B)。剔除橋分子MFG-E8、TSP-1、TSP-2中之各者降低RCS RPE對OS之吞噬(圖15C)。在一具體實施例中,RTK配體BDNF、HGF及GDNF為RCS RPE中hUTC媒介之吞噬救援所需。在另一實施例中,橋分子諸如MFG-E8、Gas6、TSP-1、及TSP-2為RCS RPE中hUTC媒介之吞噬救援所需。 Silencing of siRNA vector gene proved that BDNF, HGF, GDNF, MFG-E8, Gas6, TSP-1 and TSP-2 in hUTC were eliminated (silent). Simulated or scrambled siRNA transfection has no effect on hUTC secretion of these factors. SiRNAs targeting MFG-E8, TSP-1, TSP-2, and HGF produced almost 100% knockout efficiency; BDNF and GDNF were observed to be 80% and 65% knockouts, respectively (Figure 15A to Figure 15B). Removal of each of the bridge molecules MFG-E8, TSP-1, TSP-2 reduces the phagocytosis of OS by RCS RPE (Figure 15C). In a specific embodiment, RTK ligands BDNF, HGF and GDNF are required for phagocytosis rescue of hUTC media in RCS RPE. In another embodiment, bridge molecules such as MFG-E8, Gas6, TSP-1, and TSP-2 are required for phagocytosis rescue of hUTC media in RCS RPE.
前驅細胞諸如產後細胞亦可經基因改質以生產治療可用的基因產物,或生產用於治療腫瘤之抗癌藥物。基因改質可使用多種載體(vector)中之任一者實現,包括但不限於整合型病毒載體,例如,反轉錄病毒載體或腺相關病毒載體;非整合型複製載體,例如,乳突病毒載體、SV40載體、腺病毒載體;或複製缺陷型病毒載體。將DNA引入至細胞中之其他方法包括使用脂質體、電穿孔、粒子槍、或藉由直接DNA注射。 Precursor cells such as postpartum cells can also be genetically modified to produce therapeutically useful gene products, or to produce anti-cancer drugs for the treatment of tumors. Gene modification can be achieved using any of a variety of vectors, including but not limited to integrated viral vectors, for example, retroviral vectors or adeno-associated viral vectors; non-integrated replication vectors, for example, papillomavirus vectors , SV40 vector, adenovirus vector; or replication-defective virus vector. Other methods of introducing DNA into cells include the use of liposomes, electroporation, particle guns, or by direct DNA injection.
宿主細胞較佳係經DNA轉形或轉染,該DNA受到一或多種適當的表現控制元件諸如啟動子或增強子序列、轉錄終止子、多腺苷酸化位點等其他元件,及可選擇的標記控制或與其操作性相連。任何啟動子可用於驅動插入基因之表現。舉例而言,病毒啟動子包括但不限於CMV啟動子/增強子、SV40、乳突病毒、艾司 坦-巴爾病毒(Epstein-Barr virus)或彈性蛋白基因啟動子。在一些實施例中,用於控制關注基因之表現的控制元件可允許基因之調節表現,以使得產物僅於需要時在體內合成。若需要暫態表現,則組成性啟動子係較佳地用於非整合型及/或複製缺陷型載體中。或者,可誘導啟動子可用於當需要時驅動插入基因之表現。可誘導啟動子包括但不限於與金屬硫蛋白及熱休克蛋白質有關之啟動子。 The host cell is preferably transformed or transfected with DNA that is subjected to one or more suitable expression control elements such as promoter or enhancer sequences, transcription terminators, polyadenylation sites and other elements, and optionally Mark control or operable connection with it. Any promoter can be used to drive the performance of the inserted gene. For example, viral promoters include, but are not limited to, CMV promoter / enhancer, SV40, papillomavirus, Epstein-Barr virus, or elastin gene promoter. In some embodiments, the control elements used to control the expression of the gene of interest may allow the regulated performance of the gene so that the product is synthesized in vivo only when needed. If transient performance is required, constitutive promoter lines are preferably used in non-integrating and / or replication-defective vectors. Alternatively, inducible promoters can be used to drive the expression of inserted genes when needed. Inducible promoters include but are not limited to promoters related to metallothionein and heat shock proteins.
在引入外來DNA之後,可允許經工程化之細胞生長於濃化培養基中且隨後切換至選擇性培養基。外來DNA中之可選擇的標記賦予選擇抗性且允許細胞將例如質體上之外來DNA穩定地整合至其染色體中且生長以形成細胞群落(foci),其轉而可經選殖且擴增成細胞系。此方法可有利地用於工程化表現基因產物之細胞系。 After the introduction of foreign DNA, the engineered cells can be allowed to grow in concentrated medium and then switched to selective medium. Selectable markers in foreign DNA confer selective resistance and allow cells to stably integrate foreign DNA, such as plastids, into their chromosomes and grow to form cell communities (foci), which in turn can be colonized and expanded Into a cell line. This method can be advantageously used to engineer cell lines that express gene products.
細胞可經基因工程化以「敲除(knock out)」或「剔除(knock down)」在植入位點處促進發炎或排斥之因子之表現。以下討論用於減少靶標基因表現水準或靶標基因產物活性水準之負調控技術。如本文中所使用之「負調控(negative modulation)」係指相對於靶標基因產物在調控處理不存在下之水準及/或活性,減少該靶標基因產物之水準及/或活性。神經元或膠細胞之天然基因之表現可使用許多技術來減少或敲除,包括例如藉由使用同源重組技術使基因失活來抑制表現。一般而言,編碼蛋白質之重要區域之外顯子(或位在該區域5'側之外顯子)由陽性可選擇的標記(例如neo)中斷,防止由靶標基因生產正常mRNA且導致基因之失活。基因亦可藉由 產生基因之一部分之刪除、或藉由刪除整個基因來失活。藉由使用具有兩個與靶標基因同源之區域且該兩區域在基因體中相隔遙遠之建構體,可將在該兩個區域之間的序列刪除(Mombaerts等人,1991,Proc.Nat.Acad.Sci.U.S.A.88:3084-3087)。反義、DNA酶、核糖核酸酶、小干擾RNA(siRNA)及抑制靶標基因表現之其他此類分子亦可用於減少靶標基因活性之水準。舉例而言,抑制主要組織相容性基因複合體(HLA)表現之反義RNA分子已顯示在免疫反應方面最為多樣化。此外,三螺旋分子可被用於減少靶標基因活性之水準。這些技術由L.G.Davis等人(編),1994,BASIC METHODS IN MOLECULAR BIOLOGY,第2版,Appleton & Lange,Norwalk,CT詳細描述。 Cells can be genetically engineered to "knock out" or "knock down" the expression of factors that promote inflammation or rejection at the implantation site. The following discusses the negative regulation techniques used to reduce the target gene expression level or target gene product activity level. As used herein, "negative modulation" refers to reducing the level and / or activity of a target gene product relative to the level and / or activity of the target gene product in the absence of a modulation process. The expression of natural genes in neurons or glial cells can be reduced or knocked out using many techniques, including, for example, suppression of expression by inactivating genes using homologous recombination techniques. In general, exons (or exons located on the 5 'side of the region) that encode important regions of the protein are interrupted by a positively selectable marker (such as neo) to prevent normal mRNA production from the target gene and cause gene Deactivated. Genes can also be inactivated by creating a deletion of a part of the gene, or by deleting the entire gene. By using a construct that has two regions homologous to the target gene and the two regions are far apart in the genome, the sequence between the two regions can be deleted (Mombaerts et al., 1991, Proc. Nat. Acad. Sci. USA88: 3084-3087). Antisense, DNase, ribonuclease, small interfering RNA (siRNA) and other such molecules that inhibit the expression of target genes can also be used to reduce the level of target gene activity. For example, antisense RNA molecules that inhibit the expression of major histocompatibility gene complexes (HLA) have been shown to be the most diverse in terms of immune response. In addition, triple helix molecules can be used to reduce the level of target gene activity. These techniques are described in detail by L.G. Davis et al. (Eds.), 1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nd edition, Appleton & Lange, Norwalk, CT.
在其他態樣中,本發明提供自產後細胞製備之細胞溶解產物及細胞可溶部分,產後細胞較佳PPDC、或包含PPDC細胞之異質性或均質性細胞群、以及經基因改質或經刺激以沿著神經性路徑分化之PPDC或其族群。此類溶解產物及其部分具有許多用途。在體內使用細胞溶解產物可溶部分(亦即,實質上不含膜)例如允許將有益的細胞內環境(intracellular milieu)同種異體地用於患者,而不需引入最可能引發排斥、或其他不良免疫反應之可察覺量的細胞表面蛋白質。溶解細胞之方法為所屬技術領域中所熟知且包括機械破裂、酶破裂、或化學破裂、或其組合之各種手段。此類細胞溶解產物可由在生長培養基中之細胞直接製備,因此含有分泌的生長因子及其類似物,或可由經例如PBS或其他溶液清洗而不含培 養基之細胞製備。經清洗之細胞可以大於原始族群密度之濃度再懸浮(若較佳)。 In other aspects, the present invention provides cell lysates and cell soluble parts prepared from postpartum cells. Postpartum cells are preferably PPDC, or a heterogeneous or homogeneous cell population comprising PPDC cells, and genetically modified or stimulated Take PPDC or its ethnic groups that differentiate along a neurological path. Such lysates and their parts have many uses. The use of a soluble portion of the cell lysate in the body (ie, substantially free of membranes), for example, allows the beneficial intracellular milieu to be used allogeneically in patients without the need to introduce the most likely cause of rejection or other adverse effects A detectable amount of cell surface protein in the immune response. Methods for lysing cells are well known in the art and include various means of mechanical rupture, enzymatic rupture, or chemical rupture, or a combination thereof. Such cell lysates can be prepared directly from the cells in the growth medium, and therefore contain secreted growth factors and the like, or can be prepared from cells washed without culturing medium, such as PBS or other solutions. The washed cells can be resuspended at a concentration greater than the original population density (if preferred).
在一實施例中,全細胞溶解產物係例如藉由使細胞破裂而稍後不分離細胞部分來製備。在另一實施例中,藉由所屬技術領域中已知的常規方法,例如離心、過濾、或類似方法,將細胞膜部分與細胞之可溶部分分離。 In one embodiment, the whole cell lysate is prepared, for example, by rupturing the cells without later separating the cell parts. In another embodiment, the cell membrane portion is separated from the soluble portion of the cell by conventional methods known in the art, such as centrifugation, filtration, or the like.
由前驅細胞(諸如產後衍生細胞)群所製備之細胞溶解產物或細胞可溶部分可按原樣使用、經進一步濃縮例如藉由超濾或凍乾、或甚至乾燥後使用、經部分純化後使用、如所述技術領域中已知與醫藥上可接受之載劑或稀釋劑組合使用、或與其他化合物諸如生物製品例如醫藥上可用的蛋白質組成物組合使用。細胞溶解產物或其部分可在體外或體內單獨使用,或例如與自體或同基因活細胞一起使用。溶解產物若經引入體內,可被引入局部治療位點、或引入遠端以提供例如需要的細胞生長因子給患者。 The cell lysate or cell-soluble fraction prepared from a population of precursor cells (such as postpartum-derived cells) can be used as is, further concentrated, for example by ultrafiltration or lyophilization, or even dried, used after partial purification, As known in the technical field, it is used in combination with a pharmaceutically acceptable carrier or diluent, or in combination with other compounds such as biological products such as pharmaceutically usable protein compositions. The cell lysate or part thereof can be used alone in vitro or in vivo, or for example with autologous or syngeneic living cells. If the lysate is introduced into the body, it can be introduced into the local treatment site or distally to provide, for example, the required cell growth factor to the patient.
在進一步實施例中,產後細胞較佳PPDC可於體外培養以生產高產率之生物產物。舉例而言,此類細胞不論是天然生產受關注的特定生物產物(例如,營養因子),或經基因工程化以生產生物產物,皆可使用本文所述之培養技術選殖擴增。或者,細胞可於誘導分化成所要譜系之培養基中擴增。在任一情況下,由細胞生產且分泌至培養基中之生物產物可使用標準分離技術容易地自條件培養基分離,例如,諸如差別蛋白質沉澱、離子交換層析、膠濾層析、電泳、及HPLC等不及備舉。可使用「生物反應器(bioreactor)」 以利用流動法饋送例如體外三維培養物。基本上,在新鮮培養基通過三維培養物時,生物產物被洗出培養物且隨後可自流出物進行如上文所述之分離。 In a further embodiment, the postpartum cells, preferably PPDC, can be cultured in vitro to produce high-yield biological products. For example, whether such cells naturally produce specific biological products of interest (eg, trophic factors), or are genetically engineered to produce biological products, they can be colonized and expanded using the culture techniques described herein. Alternatively, the cells can be expanded in medium that induces differentiation into the desired lineage. In either case, biological products produced by the cells and secreted into the culture medium can be easily separated from the conditioned medium using standard separation techniques, for example, such as differential protein precipitation, ion exchange chromatography, gel filtration chromatography, electrophoresis, and HPLC, etc. Can't wait. A "bioreactor" can be used to feed, for example, an in vitro three-dimensional culture using a flow method. Basically, as fresh medium passes through the three-dimensional culture, biological products are washed out of the culture and can then be separated from the effluent as described above.
或者,關注的生物產物可保留於細胞內,因此其收集可能需要將細胞進行如上文所述之溶解。隨後可使用上文所列舉之技術中之任一或多者將生物產物純化。 Alternatively, the biological product of interest may remain within the cell, so its collection may require the cell to be lysed as described above. The biological product can then be purified using any one or more of the techniques listed above.
在另一實施例中,藉由於液體、固體或半固體基材上培養產後細胞(較佳PPDC)所生產之細胞外基質(ECM)係經製備、收集且用作為植入活細胞至需要組織修復或置換之個體中之替代物。將細胞體外培養於如本文其他地方所述之三維架構上,且培養條件將使得所要量之ECM分泌至架構上。將細胞及架構移除,且ECM係經處理以供進一步使用,例如作為可注射製劑。為實現此目的,將架構上之細胞殺死且將任何細胞碎屑自架構移除。此製程可以許多不同方式進行。舉例而言,可將活組織置於不加冷凍保存劑之液態氮中驟冷凍,或可將組織浸沒於無菌蒸餾水中以使得細胞因滲透壓爆裂。 In another embodiment, the extracellular matrix (ECM) produced by culturing postpartum cells (preferably PPDC) on a liquid, solid or semi-solid substrate is prepared, collected and used as implanted living cells to the desired tissue Replacement in the repaired or replaced entity. The cells are cultured in vitro on a three-dimensional architecture as described elsewhere herein, and the culture conditions will allow the required amount of ECM to be secreted onto the architecture. The cells and architecture are removed, and the ECM is processed for further use, for example as an injectable preparation. To achieve this, the cells on the architecture are killed and any cell debris is removed from the architecture. This process can be carried out in many different ways. For example, living tissue can be placed in liquid nitrogen without cryopreservation for rapid freezing, or the tissue can be immersed in sterile distilled water to cause the cells to burst due to osmotic pressure.
一旦細胞被殺死,細胞膜可能破裂且細胞碎屑藉由溫和清潔劑清洗諸如EDTA、CHAPS或兩性離子清潔劑處理來移除。或者,組織可經酶消化及/或用破壞細胞膜且允許移除細胞內容物之試劑萃取。此類酶之實例包括但不限於玻尿酸酶、分散酶、蛋白酶、及核酸酶。清潔劑之實例包括非離子清潔劑,諸如,例如烷芳基聚醚醇(TRITON X-100)、辛苯氧基聚乙氧基乙醇(Rohm and Haas Philadelphia,Pa.)、BRIJ-35,聚乙氧基乙醇月桂醚(Atlas Chemical Co.,San Diego,Calif.)、聚山梨醇酯20(TWEEN 20)、聚乙氧基乙醇去水山梨醇單月桂酸酯(Rohm and Haas)、聚乙烯月桂醚(Rohm and Haas);及離子清潔劑,諸如,例如十二烷基硫酸鈉、硫酸化高級脂族醇、於支鏈或非支鏈中含有7至22個碳原子之磺化烷烴及磺化烷基芳烴。 Once the cells are killed, the cell membrane may rupture and cell debris is removed by cleaning with a mild detergent such as EDTA, CHAPS, or zwitterionic detergent. Alternatively, the tissue may be digested with enzymes and / or extracted with reagents that disrupt the cell membrane and allow the removal of cell contents. Examples of such enzymes include but are not limited to hyaluronidase, dispase, protease, and nuclease. Examples of cleaning agents include nonionic cleaning agents such as, for example, alkylaryl polyether alcohols (TRITON X-100), octoxyphenoxypolyethoxyethanol (Rohm and Haas Philadelphia, Pa.), BRIJ-35, poly Ethoxyethanol lauryl ether (Atlas Chemical Co., San Diego, Calif.), Polysorbate 20 (TWEEN 20), polyethoxyethanol desorbitan monolaurate (Rohm and Haas), polyethylene Lauryl ether (Rohm and Haas); and ionic cleaners such as, for example, sodium lauryl sulfate, sulfated higher aliphatic alcohols, sulfonated alkanes containing 7 to 22 carbon atoms in branched or unbranched chains, and Sulfonated alkyl aromatics.
ECM之收集可以多種方式實現,其取決於例如新組織係形成於可生物降解或不可生物降解之三維架構上。舉例而言,若架構為不可生物降解者,則ECM可藉由使架構經歷超音波處理、高壓射水機、機械刮削、或用清潔劑或酶溫和處理、或以上之任何組合來移除。 The collection of ECM can be achieved in many ways, depending on, for example, the formation of new tissues on a biodegradable or non-biodegradable three-dimensional structure. For example, if the architecture is non-biodegradable, the ECM can be removed by subjecting the architecture to ultrasonic treatment, high-pressure water jets, mechanical scraping, or mild treatment with detergents or enzymes, or any combination of the above.
若架構為可生物降解者,則ECM可例如藉由使架構降解或溶解於溶液中來收集。或者,若可生物降解架構係由其自身可與ECM一起注射之材料組成,則可將架構及ECM全部一起處理以用於稍後注射。或者,可藉由上文所述之用於自不可生物降解架構收集ECM之任何方法,將ECM自可生物降解架構移除。所有收集製程係經較佳地設計,以使ECM不致變性。 If the framework is biodegradable, the ECM can be collected, for example, by degrading or dissolving the framework in solution. Alternatively, if the biodegradable framework is composed of materials that can be injected together with the ECM, the framework and the ECM can all be processed together for later injection. Alternatively, the ECM can be removed from the biodegradable architecture by any method described above for collecting ECM from the non-biodegradable architecture. All collection processes are better designed so that the ECM is not denatured.
在收集ECM之後,可將其進一步處理。舉例而言,可使用所述技術領域中熟知的技術諸如超音波處理,將ECM均質化成微細粒子,以使得其可通過外科針。若需要,ECM之組分可藉由γ照射來交聯。較佳地,ECM可經0.25至2百萬雷得之間照射, 以使ECM滅菌及交聯。使用具有毒性的劑諸如戊二醛之化學交聯係可能但通常不佳的。 After collecting the ECM, it can be further processed. For example, the ECM can be homogenized into fine particles using techniques well known in the technical field, such as ultrasound processing, so that it can pass through a surgical needle. If necessary, the components of ECM can be cross-linked by gamma irradiation. Preferably, the ECM can be irradiated between 0.25 and 2 million Reid to sterilize and cross-link the ECM. Chemical interactions using toxic agents such as glutaraldehyde may be but usually poor.
蛋白質諸如存在於ECM中之各種類型之膠原蛋白之量及/或比率,可藉由將本發明之細胞所生產之ECM與一或多種其他細胞型之ECM混合來調整。此外,可將生物活性物質諸如蛋白質、生長因子及/或藥品併入至ECM中。例示性生物活性物質包括組織生長因子,諸如TGF-β,及類似物,其促進注射位點之癒合及組織修復。此類額外劑可用於上文所述之任何實施例中,例如與全細胞溶解產物、可溶細胞部分、或經進一步純化的由細胞所生產之組分及產物一起利用。 The amount and / or ratio of proteins such as various types of collagen present in the ECM can be adjusted by mixing the ECM produced by the cells of the invention with one or more ECMs of other cell types. In addition, biologically active substances such as proteins, growth factors, and / or pharmaceuticals can be incorporated into the ECM. Exemplary biologically active substances include tissue growth factors, such as TGF-β, and the like, which promote healing of the injection site and tissue repair. Such additional agents can be used in any of the embodiments described above, for example with whole cell lysates, soluble cell fractions, or further purified components and products produced by cells.
在另一態樣中,本發明提供醫藥組成物,其於多種用於治療眼變性病狀之方法中使用前驅細胞諸如產後細胞(較佳PPDC)、其細胞群、由此類細胞所生產之條件培養基、及由此類細胞所生產之細胞組分及產物。某些實施例涵蓋包含活細胞(例如,單獨PPDC或PPDC與其他細胞型混合)之醫藥組成物。其他實施例涵蓋包含PPDC條件培養基之醫藥組成物。其他實施例可使用PPDC之細胞組分(例如,細胞溶解產物、可溶細胞部分、ECM、或前述任一者之組分)或產物(例如,由細胞天然生產或經由基因改質所生產之營養因子及其他生物因子、培養該些細胞之條件培養基)。在任一情況下,醫藥組成物可進一步包含其他活性劑,諸如消 炎劑、抗細胞凋亡劑、抗氧化劑、生長因子、神經營養因子或所屬技術領域中已知的神經再生、神經保護或眼科藥品。 In another aspect, the present invention provides a pharmaceutical composition that uses precursor cells such as postpartum cells (preferably PPDC), its cell population, produced by such cells in various methods for treating ocular degenerative conditions Conditioned medium, and cell components and products produced by such cells. Certain embodiments encompass pharmaceutical compositions that include living cells (eg, PPDC alone or PPDC mixed with other cell types). Other embodiments encompass pharmaceutical compositions comprising PPDC conditioned medium. Other embodiments may use cellular components of PPDC (eg, cell lysate, soluble cell fraction, ECM, or components of any of the foregoing) or products (eg, naturally produced by cells or produced by genetic modification Nutritional factors and other biological factors, conditioned medium for culturing these cells). In either case, the pharmaceutical composition may further contain other active agents, such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors, or nerve regeneration, neuroprotection, or ophthalmic drugs known in the art .
可添加至醫藥組成物之其他組分之實例包括但不限於:(1)其他神經保護或神經有益藥品;(2)選定之細胞外基質組分,諸如所屬技術領域中已知的一或多種類型的膠原蛋白、及/或生長因子、富血小板血漿、及藥品(或者,PPDC可經基因工程化以表現且生產生長因子);(3)抗細胞凋亡劑(例如,紅血球生成素(EPO)、EPO模擬體(mimetibody)、血小板生成素(thrombopoietin)、類胰島素生長因子(IGF)-I、IGF-II、肝細胞生長因子、凋亡蛋白酶抑制劑);(4)消炎化合物(例如,p38 MAP激酶抑制劑、TGF-β抑制劑、斯他汀類(statins)、IL-6及IL-1抑制劑、PEMIROLAST、TRANILAST、REMICADE、SIROLIMUS、及非固醇類消炎藥品(NSAIDS)(諸如TEPOXALIN、TOLMETIN、及SUPROFEN));(5)免疫抑制或免疫調節劑,諸如鈣調磷酸酶(calcineurin)抑制劑、mTOR抑制劑、抗增生劑、皮質類固醇及各種抗體;(6)抗氧化劑,諸如普洛布克(probucol)、維生素C與E、輔酶Q-10、麩胱甘肽、L-半胱胺酸及N-乙醯半胱胺酸;以及(6)局部麻醉劑等不及備舉。 Examples of other components that can be added to the pharmaceutical composition include, but are not limited to: (1) other neuroprotective or neurobeneficial drugs; (2) selected extracellular matrix components, such as one or more known in the art Types of collagen, and / or growth factors, platelet-rich plasma, and medicines (or, PPDC can be genetically engineered to express and produce growth factors); (3) Anti-apoptotic agents (eg, erythropoietin (EPO ), EPO mimetibody, thrombopoietin, insulin-like growth factor (IGF) -I, IGF-II, hepatocyte growth factor, apoptosis protease inhibitor); (4) anti-inflammatory compounds (for example, p38 MAP kinase inhibitors, TGF-β inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and non-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPOXALIN , TOLMETIN, and SUPROFEN)); (5) immunosuppressive or immunomodulatory agents, such as calcineurin inhibitors, mTOR inhibitors, antiproliferative agents, corticosteroids, and various antibodies; (6) antioxidants, such as Probucol ), Vitamins C and E, coenzyme Q-10, glutathione, L-cysteine and N-acetylcysteine; and (6) local anesthetics are not enough.
本發明之醫藥組成物包含前驅細胞諸如產後細胞(較佳PPDC)、由該些細胞產生之條件培養基、或其組分或產物,其與醫藥上可接受之載劑或培養基調配。合適的醫藥上可接受之載劑包括水、鹽溶液(諸如林格氏溶液)、醇、油、明膠、及碳水化合物,諸如乳糖、直鏈澱粉、或澱粉、脂肪酸酯、羥甲基纖維素、及聚乙 烯吡咯啶。此類製劑可經滅菌,且若有需要與輔助劑諸如潤滑劑、防腐劑、穩定劑、潤濕劑、乳化劑、用於影響滲透壓之鹽、緩衝劑、及著色劑混合。通常但不排他地,包含細胞組分或產物而非活細胞之醫藥組成物係經調配成液體。包含PPDC活細胞之醫藥組成物一般係經調配成液體、半固體(例如,凝膠)或固體(例如,基質、支架及類似物,以適用於眼科組織工程)。 The pharmaceutical composition of the present invention comprises precursor cells such as postpartum cells (preferably PPDC), conditioned medium produced by these cells, or a component or product thereof, which is formulated with a pharmaceutically acceptable carrier or medium. Suitable pharmaceutically acceptable carriers include water, saline solutions (such as Ringer's solution), alcohols, oils, gelatin, and carbohydrates, such as lactose, amylose, or starch, fatty acid esters, hydroxymethyl fiber Element, and polyvinylpyrrolidine. Such preparations can be sterilized and, if necessary, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, and coloring agents. Usually, but not exclusively, pharmaceutical compositions containing cellular components or products rather than living cells are formulated as liquids. Pharmaceutical compositions containing live PPDC cells are generally formulated as liquids, semi-solids (eg, gels) or solids (eg, matrices, scaffolds, and the like, suitable for ophthalmic tissue engineering).
醫藥組成物可包含醫藥化學家或生物學家所熟悉之輔助劑組分。舉例而言,其可含有在一定範圍內的抗氧化劑,該些範圍取決於所使用之抗氧化劑之種類而異。普遍使用的抗氧化劑之合理範圍為約0.01重量/體積%至約0.15重量/體積%之EDTA、約0.01重量/體積%至約2.0重量/體積%之亞硫酸鈉、及約0.01重量/體積%至約2.0重量/體積%之偏亞硫酸氫鈉。所屬技術領域中具有通常知識者可使用約0.1重量/體積%之濃度的上述各者。其他代表性化合物包括巰基丙醯基甘胺酸、N-乙醯基半胱胺酸、β-巰基乙胺、麩胱甘肽及類似物質,但亦可採用合適於眼投予之其他抗氧化劑,例如抗壞血酸及其鹽或亞硫酸鹽或偏亞硫酸氫鈉。 The pharmaceutical composition may contain auxiliary components familiar to medical chemists or biologists. For example, it may contain antioxidants in a range that varies depending on the type of antioxidant used. A reasonable range of commonly used antioxidants is about 0.01 wt / vol% to about 0.15 wt / vol% EDTA, about 0.01 wt / vol% to about 2.0 wt / vol% sodium sulfite, and about 0.01 wt / vol% to about 2.0 wt / vol% sodium metabisulfite. Those of ordinary skill in the art can use the above-mentioned concentrations at a concentration of about 0.1% by weight / volume. Other representative compounds include mercaptopropionylglycine, N-acetylcysteine, β-mercaptoethylamine, glutathione, and similar substances, but other antioxidants suitable for ocular administration may also be used , Such as ascorbic acid and its salts or sulfites or sodium metabisulfite.
可使用緩衝劑以將眼滴劑配方之pH維持在約4.0至約8.0之範圍內;以便使眼睛之刺激最小。至於直接玻璃體內或眼內注射,配方應在pH 7.2至7.5,較佳在pH 7.3至7.4。眼科組成物亦可包括適合用於向眼睛投予之張力劑。其中合適者為氯化鈉以使配方與0.9%鹽水溶液大致等滲。 Buffers can be used to maintain the pH of the eye drop formulations in the range of about 4.0 to about 8.0; in order to minimize eye irritation. For direct intravitreal or intraocular injection, the formulation should be at pH 7.2 to 7.5, preferably at pH 7.3 to 7.4. The ophthalmic composition may also include tonicity agents suitable for administration to the eye. The suitable one is sodium chloride to make the formula approximately isotonic with 0.9% saline solution.
在某些實施例中,醫藥組成物係與黏度增強劑一起調配。例示性劑為羥乙基纖維素、羥丙基纖維素、甲基纖維素、及聚乙烯吡咯啶酮。若需要,醫藥組成物可添加共溶劑。合適的共溶劑可包括甘油、聚乙二醇(PEG)、聚山梨醇酯、丙二醇、及聚乙烯醇。亦可包括防腐劑,例如,氯化烷基二甲基苄基銨、氯化苯釷、氯丁醇、乙酸苯汞、硝酸苯汞、硫柳汞、對羥苄酸甲酯、或對羥苄酸丙酯。 In some embodiments, the pharmaceutical composition is formulated with a viscosity enhancer. Exemplary agents are hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, and polyvinylpyrrolidone. If necessary, a co-solvent can be added to the pharmaceutical composition. Suitable co-solvents may include glycerin, polyethylene glycol (PEG), polysorbate, propylene glycol, and polyvinyl alcohol. May also include preservatives, for example, alkyl dimethyl benzyl ammonium chloride, phenyl thorium chloride, chlorobutanol, phenylmercuric acetate, phenylmercuric nitrate, thimerosal, methyl paraben, or paraben Propyl ester.
用於注射之配方較佳經設計用於單次使用投予且不含有防腐劑。可注射溶液應具有等於0.9%氯化鈉溶液(290至300毫滲莫耳之滲透壓)之等滲度。此可藉由添加氯化鈉或如上文所列舉之其他共溶劑、或如上文所列舉之賦形劑諸如緩衝劑及抗氧化劑獲得。 The formulation for injection is preferably designed for single-use administration and does not contain preservatives. The injectable solution should have an isotonicity equal to 0.9% sodium chloride solution (290 to 300 mOsmol). This can be obtained by adding sodium chloride or other co-solvents as listed above, or excipients such as buffers and antioxidants as listed above.
眼前房之組織浸浴於前房液中,而視網膜係持續暴露於玻璃體。這些流體/凝膠以高度還原之氧化還原狀態存在,因為其含有抗氧化劑化合物及酶。因此,眼科組成物中包括還原劑可為有利的。合適的還原劑包括N-乙醯半胱胺酸、抗壞血酸或鹽形式、及亞硫酸鈉或偏亞硫酸氫鈉,其中抗壞血酸及/或N-乙醯半胱胺酸或麩胱甘肽尤其適合用於可注射溶液。 The tissue in the anterior chamber of the eye is bathed in the anterior chamber fluid, while the retina is continuously exposed to the vitreous. These fluids / gels exist in a highly reduced redox state because they contain antioxidant compounds and enzymes. Therefore, it may be advantageous to include a reducing agent in the ophthalmic composition. Suitable reducing agents include N-acetylcysteine, ascorbic acid or salt forms, and sodium sulfite or sodium metabisulfite, wherein ascorbic acid and / or N-acetylcysteine or glutathione are particularly suitable for use in Injectable solution.
包含細胞或條件培養基、或細胞組分或細胞產物之醫藥組成物可以所屬技術領域中已知的數種遞送方式中之一或多者遞送至患者眼睛。在可適合用於一些情況中之一個實施例中,組成物以眼滴劑或洗眼劑之形式局部遞送至眼睛。在另一實施例中,組成 物可經由週期性眼內注射或藉由輸注於灌洗溶液諸如BSS或BSS PLUS(Alcon USA,Fort Worth,Tex.)中遞送至眼睛內之各種位置。或者,組成物可以所屬技術領域中具有通常知識者已知的其他眼科劑量形式施加,諸如預形成或原位形成凝膠或脂質體,例如Herrero-Vanrell之美國專利第5,718,922號中所揭露。在另一實施例中,組成物可經由隱形眼鏡(例如Lidofilcon B,Bausch & Lomb CW79或DELTACON(Deltafilcon A))或其他暫時停留在眼睛表面上的物體遞送至或遞送通過需要治療之眼睛之水晶體。在其他實施例中,可採用諸如膠原蛋白角膜護罩(例如BIO-COR可溶性角膜護罩,Summit Technology,Watertown,Mass.)之支持物。組成物亦可藉由輸注至眼球中來投予,該輸注係經由滲透泵(ALZET,Alza Corp.,Palo Alto,Calif.)之套管或藉由植入定時釋放膠囊(OCCUSENT)或可生物降解盤(OCULEX,OCUSERT)之任一者進行。此等投予途徑具有向眼睛提供醫藥組成物之連續供應之優點。此可有利於角膜之局部遞送。 The pharmaceutical composition comprising cells or conditioned medium, or cell components or cell products can be delivered to the patient's eye by one or more of several delivery methods known in the art. In one embodiment that may be suitable for use in some situations, the composition is delivered locally to the eye in the form of eye drops or eye wash. In another embodiment, the composition can be delivered to various locations in the eye via periodic intraocular injection or by infusion in a lavage solution such as BSS or BSS PLUS (Alcon USA, Fort Worth, Tex.). Alternatively, the composition may be applied in other ophthalmic dosage forms known to those of ordinary skill in the art, such as preformed or in situ formed gels or liposomes, such as disclosed in US Patent No. 5,718,922 to Herrero-Vanrell. In another embodiment, the composition may be delivered to or through the lens of the eye in need of treatment via contact lenses (eg Lidofilcon B, Bausch & Lomb CW79 or DELTACON (Deltafilcon A)) or other objects temporarily resting on the surface of the eye . In other embodiments, supports such as collagen corneal shields (eg BIO-COR soluble corneal shields, Summit Technology, Watertown, Mass.) May be used. The composition can also be administered by infusion into the eyeball, the infusion is through a cannula of an osmotic pump (ALZET, Alza Corp., Palo Alto, Calif.) Or by implantation of a time-release capsule (OCCUSENT) or biological Degradation disk (OCULEX, OCUSERT) any one. These routes of administration have the advantage of providing a continuous supply of pharmaceutical composition to the eye. This may facilitate local delivery of the cornea.
包含於半固體或固體載劑中之活細胞之醫藥組成物一般經調配用於外科植入眼損傷或痛苦之部位。應理解,液體組成物亦可藉由外科程序投予,例如條件培養基。在具體實施例中,半固體或固體醫藥組成物可包含半透性凝膠、格架(lattice)、細胞支架及類似物,其可為不可生物降解者或可生物降解者。舉例而言,在某些實施例中,使外源性細胞與其周圍環境隔絕,但仍使該些細胞得以分泌且遞送生物分子至周圍細胞可為合意的或適當的。在這些 實施例中,細胞可經調配成包含活的PPDC或含PPDC之細胞群之自主性植入物(autonomous implant),該等活的PPDC或含PPDC之細胞群係由不可降解、選擇性可滲透之阻障圍繞以物理性分離移植細胞與宿主組織。此類植入物有時稱為「免疫保護性(immunoprotective)」,因為其具有防止免疫細胞及巨分子在醫藥誘導免疫抑制不存在下殺死移植細胞之能力(有關此類裝置及方法之綜述,參見例如P.A.Tresco等人,2000,Adv.Drug Delivery Rev.42:3-27)。 The pharmaceutical composition of living cells contained in a semi-solid or solid carrier is generally formulated for surgical implantation at the site of eye injury or pain. It should be understood that liquid compositions can also be administered by surgical procedures, such as conditioned medium. In a specific embodiment, the semi-solid or solid pharmaceutical composition may include a semi-permeable gel, lattice, cell scaffold, and the like, which may be non-biodegradable or biodegradable. For example, in certain embodiments, it may be desirable or appropriate to isolate exogenous cells from their surrounding environment, but still allow these cells to be secreted and deliver biomolecules to surrounding cells. In these embodiments, the cells can be formulated into autonomous implants containing live PPDC or PPDC-containing cell populations, which are made of non-degradable, selective The permeable barrier surrounds the physical separation of transplanted cells and host tissue. Such implants are sometimes referred to as "immunoprotective" because of their ability to prevent immune cells and macromolecules from killing transplanted cells in the absence of medically induced immunosuppression (a review of such devices and methods) , See, for example, PATresco et al., 2000, Adv. Drug Delivery Rev. 42: 3-27).
在其他實施例中,不同種類之可降解凝膠及網狀物(network)可用於本發明之醫藥組成物。舉例而言,尤其合適於持續釋放配方之可降解材料包括生物相容性聚合物,諸如聚(乳酸)、聚(乳-共-乙醇酸)、甲基纖維素、玻尿酸、膠原蛋白、及類似物。藥品遞送媒劑中可降解聚合物之結構、選擇、及用途已綜述於數個出版物中,包括A.Domb等人,1992,Polymers for Advanced Technologies 3:279-291。Wong等人之美國專利第5,869,079號揭示可生物降解持續釋放眼植入物中親水性與疏水性實體之組合。此外,Guo等人之美國專利第6,375,972號、Chen等人之美國專利第5,902,598號、Wong等人之美國專利第6,331,313號、Ogura等人之美國專利第5,707,643號、Weiner等人之美國專利第5,466,233號、及Avery等人之美國專利第6,251,090號各描述可用於遞送醫藥組成物之眼內植入物裝置及系統。 In other embodiments, different types of degradable gels and networks can be used in the pharmaceutical composition of the present invention. For example, biodegradable materials that are particularly suitable for sustained release formulations include biocompatible polymers such as poly (lactic acid), poly (milk-co-glycolic acid), methyl cellulose, hyaluronic acid, collagen, and the like Thing. The structure, selection, and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al., 1992, Polymers for Advanced Technologies 3: 279-291. US Patent No. 5,869,079 to Wong et al. Discloses a combination of biodegradable sustained release hydrophilic and hydrophobic entities in ocular implants. In addition, US Patent No. 6,375,972 by Guo et al., US Patent No. 5,902,598 by Chen et al., US Patent No. 6,331,313 by Wong et al., US Patent No. 5,707,643 by Ogura et al., US Patent No. 5,466,233 by Weiner et al. No. 6, and Avery et al. US Patent No. 6,251,090 each describe an intraocular implant device and system that can be used to deliver pharmaceutical compositions.
在例如用於修復神經病灶諸如損傷或切斷之視神經之其他實施例中,遞送於可生物降解(較佳生物可再吸收或生物可吸收)支架或基質之上或之中的細胞可為合意的或適當的。這些一般三維生物材料含有附著至支架、分散於支架內、或併入包埋於支架中之細胞外基質中之活細胞。一旦植入至身體之目標區域中,這些植入物變得與宿主組織整合,其中移植細胞逐漸變得確立(參見例如P.A.Tresco等人,2000,見前文;亦參見D.W.Hutmacher,2001,J.Biomater.Sci.Polymer Edn.12:107-174)。 In other embodiments, such as for repairing neurological lesions such as damaged or severed optic nerves, cells delivered on or in a biodegradable (preferably bioresorbable or bioresorbable) scaffold or matrix may be desirable Or appropriate. These general three-dimensional biomaterials contain living cells attached to the scaffold, dispersed within the scaffold, or incorporated into the extracellular matrix embedded in the scaffold. Once implanted into the target area of the body, these implants become integrated with the host tissue, where the transplanted cells gradually become established (see, for example, PATresco et al., 2000, see the foregoing; see also DWHutmacher, 2001, J. Biomater. Sci. Polymer Edn. 12: 107-174).
可用於本發明中之支架或基質(有時統稱為「架構(framework)」)材料之實例包括非織物墊、多孔發泡體、或自組裝肽。非織物墊可例如使用包含乙醇酸及乳酸(PGA/PLA)之合成可吸收共聚物之纖維形成,其以商品名VICRYL(Ethicon,Inc.,Somerville,N.J)銷售。亦可利用由例如聚(ε-己內酯)/聚(乙醇酸)(PCL/PGA)共聚物組成之發泡體,其藉由諸如冷凍乾燥、或凍乾之製程形成,如美國專利第6,355,699號中所討論。亦可使用諸如自組裝肽(例如,RAD16)之水凝膠。原位形成之可降解網狀物亦適合用於本發明(參見例如Anseth,K.S.等人,2002,J.Controlled Release 78:199-209;Wang,D.等人,2003,Biomaterials 24:3969-3980;He等人之美國專利公開案2002/0022676)。這些材料係經調配成適用於注射之流體,且隨後可藉由多種手段(例如,改變溫度、pH、暴露於光)誘導以原位或體內形成可降解水凝膠網狀物。 Examples of scaffold or matrix (sometimes collectively referred to as "framework") materials that can be used in the present invention include non-woven mats, porous foams, or self-assembling peptides. Non-woven mats can be formed, for example, using fibers containing synthetic absorbable copolymers of glycolic acid and lactic acid (PGA / PLA), which are sold under the trade name VICRYL (Ethicon, Inc., Somerville, N.J). It is also possible to use a foam composed of, for example, poly (ε-caprolactone) / poly (glycolic acid) (PCL / PGA) copolymer, which is formed by a process such as freeze drying or freeze drying, as described in Discussed in 6,355,699. Hydrogels such as self-assembling peptides (eg, RAD16) can also be used. Degradable webs formed in situ are also suitable for use in the present invention (see, for example, Anseth, KS et al., 2002, J. Controlled Release 78: 199-209; Wang, D. et al., 2003, Biomaterials 24: 3969- 3980; He et al. US Patent Publication 2002/0022676). These materials are formulated into fluids suitable for injection, and can then be induced to form a degradable hydrogel network in situ or in vivo by various means (eg, changing temperature, pH, exposure to light).
在另一實施例中,架構為毛氈(felt),其可由自生物可吸收材料(例如,PGA、PLA、PCL共聚物或摻合物、或玻尿酸)製成之多絲纖維紗(multifilament yarn)組成。使用由捲曲、裁剪、分梳及針刺所組成之標準紡織加工技術將紗製成毛氈。在另一實施例中,細胞係經接種至可為複合結構之發泡體支架上。 In another embodiment, the framework is a felt, which may be multifilament yarn made from bioabsorbable materials (eg, PGA, PLA, PCL copolymer or blend, or hyaluronic acid) composition. The yarn is made into felt using standard textile processing techniques consisting of crimping, cutting, carding and needle punching. In another embodiment, the cell line is seeded onto a foam scaffold that can be a composite structure.
在許多上文所提及之實施例中,可將架構模製成有用的形狀。此外應理解,可將PPDC培養於預形成、不可降解之外科或可植入裝置上,該培養方式為例如對應於用於製備例如含纖維母細胞GDC血管內線圈之方式(Marx,W.F.等人,2001,Am.J.Neuroradiol.22:323-333)。 In many of the embodiments mentioned above, the framework can be molded into useful shapes. In addition, it should be understood that PPDC can be cultured on a pre-formed, non-degradable surgical or implantable device, which corresponds, for example, to the method used to prepare, for example, fibroblast-containing GDC intravascular coils (Marx, WF et al. , 2001, Am. J. Neuroradiol. 22: 323-333).
在接種細胞之前,基質、支架或裝置可經處理以增強細胞附著。舉例而言,在接種之前,尼龍基質可用0.1莫耳乙酸處理且培養於聚離胺酸、PBS、及/或膠原蛋白中以塗布尼龍。聚苯乙烯可使用硫酸類似地處理。架構之外表面亦可經改質以改善細胞之附著或生長及組織之分化,諸如藉由血漿塗布架構或添加一或多種蛋白質(例如,膠原蛋白、彈性纖維、網狀纖維)、醣蛋白、醣胺聚醣(例如,硫酸肝素、軟骨素-4-硫酸鹽、軟骨素-6-硫酸鹽、硫酸皮膚素、硫酸角蛋白)、細胞基質及/或其他材料,諸如但不限於明膠、藻酸鹽、瓊脂、瓊脂糖、及植物膠等其他者。 Prior to seeding cells, the matrix, scaffold or device can be treated to enhance cell attachment. For example, before inoculation, the nylon matrix may be treated with 0.1 mol acetic acid and cultured in polyamic acid, PBS, and / or collagen to coat nylon. Polystyrene can be similarly treated using sulfuric acid. The outer surface of the framework can also be modified to improve cell attachment or growth and tissue differentiation, such as by coating the framework with plasma or adding one or more proteins (eg, collagen, elastic fibers, mesh fibers), glycoproteins, Glycosaminoglycans (eg, heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), cell matrix, and / or other materials such as but not limited to gelatin, algae Salts, agar, agarose, vegetable gums and others.
含有活細胞之架構係根據所屬技術領域中已知的方法製備。舉例而言,細胞可自由生長於培養容器中至次長滿或長滿、自培養物剝離(lifted)且接種至架構上。若需要,可在接種細胞之前、 之期間、或之後,將生長因子添加至培養基以引發分化及組織形成。或者,架構本身可經改質以使得其上之細胞生長增強,或使得排斥植入物之風險減少。因此,可將一或多種生物活性化合物包括但不限於消炎劑、免疫抑制劑或生長因子添加至架構以用於局部釋放。 The framework containing living cells is prepared according to methods known in the art. For example, cells can grow freely in a culture vessel until they are overgrown or overgrown, lifted from the culture, and seeded onto the framework. If necessary, growth factors can be added to the medium before, during, or after seeding the cells to induce differentiation and tissue formation. Alternatively, the architecture itself can be modified to enhance the growth of cells on it, or to reduce the risk of repelling the implant. Therefore, one or more biologically active compounds, including but not limited to anti-inflammatory agents, immunosuppressive agents, or growth factors can be added to the framework for local release.
前驅細胞諸如產後細胞(較佳hUTC或PDC)、或其細胞群、或由此類細胞所生產之條件培養基或其他組分或產物可以多種方式使用以支持且有利眼細胞及組織之修復及再生。此類用途涵蓋體外、離體及體內方法。以下所闡述之方法係關於PPDC,但其他產後細胞亦可適用於該些方法中。 Precursor cells such as postnatal cells (preferably hUTC or PDC), or their cell populations, or conditioned medium or other components or products produced by such cells can be used in a variety of ways to support and facilitate the repair and regeneration of eye cells and tissues . Such uses include in vitro, ex vivo, and in vivo methods. The methods described below are related to PPDC, but other postpartum cells can also be applied to these methods.
在一實施例中,前驅細胞諸如產後細胞(較佳hUTC或PDC)、及由其產生之條件培養基可於體外使用以篩選多種化合物之有效性及醫藥劑、生長因子、調節因子及類似物之細胞毒性。舉例而言,此類篩選可於實質上均質性PPDC族群上進行以評估欲與PPDC一起調配或共同投予以用於治療眼病症之候選化合物之效力或毒性。或者,出於評估新醫藥藥品候選者之效力之目的,此類篩選可於經刺激以分化成眼睛中所發現之細胞型之PPDC、或其前驅上進行。在此實施例中,PPDC係維持在體外並暴露於待測試之 化合物。潛在細胞毒性化合物之活性可藉由其損傷或殺死培養物中之細胞的能力來量測。此可藉由活體染色技術容易地評估。 In one embodiment, precursor cells such as postpartum cells (preferably hUTC or PDC), and the conditioned medium produced therefrom can be used in vitro to screen the effectiveness of various compounds and pharmaceutical agents, growth factors, regulatory factors and the like Cytotoxicity. For example, such screening can be performed on a substantially homogeneous PPDC population to assess the efficacy or toxicity of candidate compounds to be formulated with PPDC or co-administered for the treatment of eye disorders. Alternatively, for the purpose of evaluating the effectiveness of new pharmaceutical drug candidates, such screening may be performed on PPDCs stimulated to differentiate into the cell types found in the eye, or their precursors. In this example, PPDC was maintained in vitro and exposed to the compound to be tested. The activity of potentially cytotoxic compounds can be measured by their ability to damage or kill cells in culture. This can be easily assessed by in vivo staining techniques.
如上文所討論,PPDC可於體外培養以生產由細胞自然生產、或由細胞在經誘導以分化成其他譜系時所生產、或由細胞經由基因改質所生產之生物產物。舉例而言,發現TIMP1、TPO、KGF、HGF、FGF、HBEGF、BDNF、MIP1b、MCP1、RANTES、I309、TARC、MDC與IL-8皆分泌自生長於生長培養基中之臍衍生細胞。發現TIMP1、TPO、KGF、HGF、HBEGF、BDNF、MIP1a、MCP-1、RANTES、TARC、伊紅趨素(Eotaxin)、與IL-8皆分泌自培養於生長培養基中之胎盤衍生PPDC(參見實例)。 As discussed above, PPDC can be cultured in vitro to produce biological products that are produced naturally by cells, or when cells are induced to differentiate into other lineages, or by genetic modification of cells. For example, TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1, RANTES, I309, TARC, MDC, and IL-8 were found to be secreted from umbilical-derived cells grown in growth medium. It was found that TIMP1, TPO, KGF, HGF, HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 are all secreted from placenta-derived PPDC cultured in growth medium (see examples ).
就此而言,本發明之實施例之特徵在於使用PPDC以生產條件培養基。自PPDC生產條件培養基可來自未分化之PPDC或來自在刺激分化之條件下培養之PPDC。此類條件培養基係經考慮用於例如上皮或神經前體細胞之體外或離體培養,或用於體內以支持包含PPDC之均質性族群或包含PPDC及其他前驅之異質性族群之移植細胞。 In this regard, embodiments of the present invention are characterized by the use of PPDC to produce conditioned medium. The conditioned medium produced from PPDC can be derived from undifferentiated PPDC or from PPDC cultured under conditions that stimulate differentiation. Such conditioned media are considered for use in, for example, in vitro or ex vivo culture of epithelial or neural precursor cells, or in vivo to support transplanted cells of a homogeneous population comprising PPDC or a heterogeneous population comprising PPDC and other precursors.
出於多種目的可使用PPDC之細胞溶解產物、可溶細胞部分或組分、或其ECM或組分。如上文所提及,此等組分中之一些可用於醫藥組成物中。在其他實施例中,細胞溶解產物或ECM係用於塗布或以其他方式處理欲用於外科、或用於植入、或用於離體目的之物質或裝置,以促進在此類治療程序中所接觸之細胞或組織之癒合或生存。 Cell lysates, soluble cell parts or components of PPDC, or their ECMs or components can be used for a variety of purposes. As mentioned above, some of these components can be used in pharmaceutical compositions. In other embodiments, the cell lysate or ECM is used to coat or otherwise treat substances or devices intended for surgery, or for implantation, or for ex vivo purposes to facilitate in such treatment procedures Healing or survival of the contacted cells or tissues.
如實例22及24中所述,已證明當PPDC與成體神經前驅細胞共培養生長時,PPDC具有支持該些細胞生存、生長及分化之能力。同樣,先前研究指出,PPDC可經由營養機制發揮支持視網膜細胞之功能。(US 2010-0272803)。因此,PPDC有利地用於體外共培養物中以提供營養支持給其他細胞,尤其神經細胞及神經和眼前驅(例如,神經幹細胞及視網膜或角膜上皮幹細胞)。至於共培養,將PPDC與所要其他細胞在兩種細胞型相接觸之條件下共培養可為合意的。此可例如藉由將細胞以異質性細胞群之形式接種於培養基中或接種至合適的培養基材上來達成。或者,首先可使PPDC生長至長滿,且隨後充當培養物中第二所要細胞型之基材。在此後者之實施例中,在共培養時期之後,可例如藉由膜或類似裝置將細胞進一步物理分離,以使得該其他細胞型可經移除且單獨使用。使用PPDC共培養以促進神經或眼細胞型之擴增及分化可具有在研究及臨床/治療領域中之適用性。舉例而言,可利用PPDC共培養以利培養物中之此類細胞之生長及分化,例如為了基礎研究目的或用於藥品篩選檢定。PPDC共培養亦可用於神經或眼前驅之離體擴增,以供稍後投予用於治療目的。舉例而言,神經或眼前驅細胞可自個體收集、與PPDC共培養離體擴增、隨後返回至該個體(自體轉移)或另一個體(同基因或同種異體轉移)。在此等實施例中,應理解在離體擴增之後,可將包含PPDC及前驅之混合細胞群向需要治療之患者投予。或者,在自體轉移為適當的或合意的情況下,可將共培 養細胞群在培養物中物理分離,使得能夠移除自體前驅以用於向患者投予。 As described in Examples 22 and 24, when PPDC is grown in co-culture with adult neural precursor cells, PPDC has the ability to support the survival, growth and differentiation of these cells. Similarly, previous studies have pointed out that PPDC can play a role in supporting retinal cells through nutritional mechanisms. (US 2010-0272803). Therefore, PPDC is advantageously used in in vitro co-cultures to provide nutritional support to other cells, especially nerve cells and nerve and eye precursors (eg, neural stem cells and retinal or corneal epithelial stem cells). As for the co-cultivation, it may be desirable to co-culture the PPDC with the desired other cells under conditions in which the two cell types are in contact. This can be achieved, for example, by seeding the cells in a medium in the form of a heterogeneous cell population or onto a suitable culture substrate. Alternatively, the PPDC can first be grown to fullness and then serve as a substrate for the second desired cell type in culture. In this latter embodiment, after the co-cultivation period, the cells can be further physically separated, such as by a membrane or similar device, so that the other cell types can be removed and used alone. The use of PPDC co-culture to promote the expansion and differentiation of neural or ocular cell types may have applicability in research and clinical / therapeutic fields. For example, PPDC co-culture can be used to facilitate the growth and differentiation of such cells in culture, for example for basic research purposes or for drug screening tests. PPDC co-culture can also be used for ex vivo expansion of nerve or eye precursors for later administration for therapeutic purposes. For example, nerve or ocular precursor cells can be collected from an individual, co-cultured with PPDC, expanded in vitro, and then returned to the individual (autologous transfer) or another entity (syngeneic or allogeneic transfer). In these embodiments, it should be understood that after ex vivo expansion, a mixed cell population comprising PPDC and precursors can be administered to patients in need of treatment. Alternatively, where autologous transfer is appropriate or desirable, the co-cultured cell population can be physically separated in the culture, allowing the autologous precursor to be removed for administration to the patient.
如實例中所闡述,條件培養基可有效地用於治療眼變性病狀。一旦移植至眼睛中之目標位置中,來自前驅細胞(諸如PPDC)之條件培養基原位提供營養支持給眼細胞。 As illustrated in the examples, conditioned medium can be effectively used to treat ocular degenerative conditions. Once transplanted into the target location in the eye, conditioned medium from precursor cells (such as PPDC) provides nutritional support to the eye cells in situ.
來自前驅細胞(諸如PPDC)之條件培養基可與以下一起投予:其他有益的藥品、生物分子(諸如生長因子、營養因子)、條件培養基(來自前驅細胞或分化細胞培養物)、或其他活性劑(諸如消炎劑、抗細胞凋亡劑、抗氧化劑、生長因子、神經營養因子或如所屬技術領域中已知的神經再生或神經保護藥品)。當條件培養基與其他藥劑投予時,其可以單一醫藥組成物之形式一起投予,或與其他劑同時或依次(在投予其他劑之前或之後)投予之分開醫藥組成物。 Conditioned medium from precursor cells (such as PPDC) can be administered together with: other beneficial drugs, biomolecules (such as growth factors, trophic factors), conditioned medium (from precursor cells or differentiated cell cultures), or other active agents (Such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors or nerve regeneration or neuroprotective drugs as known in the art). When the conditioned medium is administered with other agents, it can be administered together in the form of a single pharmaceutical composition, or separately or simultaneously with other agents (before or after the administration of other agents).
可與前驅細胞(諸如PPDC)及條件培養基產物一起投予之其他組分之實例包括但不限於:(1)其他神經保護或神經有益藥品;(2)選定之細胞外基質組分,諸如所屬技術領域中已知的一或多種類型的膠原蛋白、及/或生長因子、富血小板血漿、及藥品(或者,細胞可經基因工程化以表現且生產生長因子);(3)抗細胞凋亡劑(例如,紅血球生成素(EPO)、EPO模擬體(mimetibody)、血小板生成素(thrombopoietin)、類胰島素生長因子(IGF)-I、IGF-II、肝細 胞生長因子、凋亡蛋白酶抑制劑);(4)消炎化合物(例如,p38 MAP激酶抑制劑、TGF-β抑制劑、斯他汀類(statins)、IL-6及IL-I抑制劑、PEMIROLAST、TRANILAST、REMICADE、SIROLIMUS、及非固醇類消炎藥品(NSAIDS)(諸如TEPOXALIN、TOLMETIN、及SUPROFEN));(5)免疫抑制或免疫調節劑,諸如鈣調磷酸酶(calcineurin)抑制劑、mTOR抑制劑、抗增生劑、皮質類固醇及各種抗體;(6)抗氧化劑,諸如普洛布克(probucol)、維生素C與E、輔酶Q-10、麩胱甘肽、L-半胱胺酸及N-乙醯半胱胺酸;以及(6)局部麻醉劑等不及備舉。 Examples of other components that can be administered with precursor cells (such as PPDC) and conditioned medium products include, but are not limited to: (1) other neuroprotective or neurobeneficial drugs; (2) selected extracellular matrix components, such as One or more types of collagen, and / or growth factors, platelet-rich plasma, and medicines known in the technical field (or, cells can be genetically engineered to express and produce growth factors); (3) Anti-apoptosis Agents (eg, erythropoietin (EPO), EPO mimetibody, thrombopoietin, insulin-like growth factor (IGF) -I, IGF-II, hepatocyte growth factor, apoptosis protease inhibitor) ; (4) Anti-inflammatory compounds (eg, p38 MAP kinase inhibitors, TGF-β inhibitors, statins, IL-6 and IL-I inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and non-sterols Anti-inflammatory drugs (NSAIDS) (such as TEPOXALIN, TOLMETIN, and SUPROFEN); (5) Immunosuppressive or immunomodulatory agents, such as calcineurin inhibitors, mTOR inhibitors, antiproliferative agents, corticosteroids and various Antibody; (6) Antioxidants, such as probucol, vitamins C and E, coenzyme Q-10, glutathione, L-cysteine and N-acetylcysteine; and (6) local anesthetics, etc. Can't wait.
液體或流體醫藥組成物可經投予至眼睛中更一般的位置(例如,局部地或眼內地)。 The liquid or fluid pharmaceutical composition can be administered to a more general location in the eye (eg, locally or intraocularly).
其他實施例涵蓋藉由投予醫藥組成物來治療眼變性病狀之方法,該些醫藥組成物包含來自前驅細胞(諸如PPDC)之條件培養基、或由該些細胞天然生產或經由細胞之基因改質生產之營養因子及其他生物因子。此外,這些方法可進一步包含投予其他活性劑,諸如生長因子、神經營養因子或如所屬技術領域中已知的神經再生或神經保護藥品。 Other embodiments encompass methods for treating ocular degenerative conditions by administering pharmaceutical compositions that include conditioned medium from precursor cells (such as PPDC), or are naturally produced by these cells or genetically modified by the cells Nutrients and other biological factors for quality production. In addition, these methods may further comprise the administration of other active agents, such as growth factors, neurotrophic factors, or nerve regeneration or neuroprotective drugs as known in the art.
用於投予來自前驅細胞(諸如PPDC)之條件培養基、或本文所述之任何其他醫藥組成物之劑量形式及方案係根據良好的醫療實踐得以發展,其考慮到個別患者之條件,例如,眼變性病狀之性質與程度、年齡、性別、體重及一般醫學病狀、及醫學從業者 已知的其他因素。因此,欲向患者投予之醫藥組成物之有效量係藉由如所屬技術領域中已知的這些考量判定。 The dosage forms and protocols for the administration of conditioned medium from precursor cells (such as PPDC), or any other pharmaceutical composition described herein, are developed according to good medical practice, taking into account the conditions of individual patients, for example, ocular The nature and degree of degenerative conditions, age, gender, weight and general medical conditions, and other factors known to medical practitioners. Therefore, the effective amount of the pharmaceutical composition to be administered to the patient is determined by these considerations as known in the art.
在開始細胞治療之前,以醫藥免疫抑制患者可為合意的或適當的。此可經由使用全身或局部免疫抑制劑來實現,或可藉由遞送於囊封裝置中之細胞來實現,如上文所述。這些及其他用於減少或消除對移植細胞之免疫反應之手段為所屬技術領域中已知的。替代地,條件培養基可自經基因改質以減少其免疫原性(immunogenicity)之PPDC製備,如上文所提及。 Before starting cell therapy, it may be desirable or appropriate to suppress the patient with medical immunosuppression. This can be achieved by using systemic or local immunosuppressants, or can be achieved by cells delivered in an encapsulated device, as described above. These and other means for reducing or eliminating the immune response to transplanted cells are known in the art. Alternatively, the conditioned medium may be prepared from PPDC that has been genetically modified to reduce its immunogenicity, as mentioned above.
移植細胞於活體患者中之生存可經由使用多種掃描技術來判定,例如,電腦化斷層(CAT或CT)掃描、磁共振成像(MRI)或正電子發射斷層攝影(PET)掃描。判定移植之生存亦可藉由移除組織且目視或經由顯微鏡檢查該組織來進行事後分析。或者,細胞可用對神經或眼細胞或其產物(例如,神經傳遞物)具特異性之染色劑處理。移植細胞亦可藉由事先併入示蹤染料諸如玫瑰紅或螢光素標記微球、快藍(fast blue)、鐵微粒、雙苯甲醯胺或基因引入之報告基因產物,諸如β-半乳糖苷酶或β-葡萄醣醛酸酶來識別。 The survival of transplanted cells in living patients can be determined by using various scanning techniques, for example, computerized tomography (CAT or CT) scanning, magnetic resonance imaging (MRI) or positron emission tomography (PET) scanning. The survival of the transplant can also be determined by removing the tissue and visually or by examining the tissue through a microscope for post-mortem analysis. Alternatively, the cells can be treated with stains specific for nerve or eye cells or their products (eg, neurotransmitters). Transplanted cells can also be obtained by incorporating tracer dyes such as rose red or luciferin-labeled microspheres, fast blue, iron particles, bisbenzamide, or gene-introduced reporter gene products, such as Lactosidase or β-glucuronidase to recognize.
將移植細胞或條件培養基功能性整合至眼組織或個體中可藉由檢查受損或患病之眼功能的恢復來評估。舉例而言,治療黃斑變性或其他視網膜病變之有效性可藉由視覺銳度之改善及評估立體眼底彩色相片之異常及分級來判定。(年齡相關性眼睛疾病研究調查小組,NEI,NIH,AREDS報告第8號,2001,Arch.Ophthalmol.119:1417-1436)。 The functional integration of transplanted cells or conditioned medium into ocular tissues or individuals can be assessed by examining the recovery of damaged or diseased eye function. For example, the effectiveness of treating macular degeneration or other retinopathy can be judged by the improvement of visual acuity and the evaluation of abnormalities and grading of stereoscopic fundus color photos. (Age-related eye disease research investigation team, NEI, NIH, AREDS Report No. 8, 2001, Arch. Ophthalmol. 119: 1417-1436).
在另一態樣中,本發明提供套組,其於多種如上所述用於眼再生及修復之方法中利用前驅細胞(諸如PPDC)、及細胞群、由該些細胞(較佳PPDC)所製備之條件培養基及其組分及產物。當用於治療眼變性病狀、或其他計劃治療時,該些套組可包括一或多種細胞群或條件培養基(包括至少產後細胞或衍生自產後細胞之條件培養基)、及醫藥上可接受之載劑(液體、半固體或固體)。套組亦可選地可包括例如藉由注射投予細胞及條件培養基之手段。套組進一步可包括使用細胞及條件培養基之說明書。經製備用於野外醫院用途,諸如軍事用途之套組可包括全程序供應(full-procedure supply),其包括組織支架、手術用縫線、及類似物,其中細胞或條件培養基與急性受傷之修復結合使用。如本文所述之用於檢定及體外方法之套組可含有例如以下中之一或多者:(1)PPDC或其組分、或PPDC之條件培養基或其他產物;(2)用於實踐體外方法之試劑;(3)適當時選用的其他細胞或細胞群;及(4)用於指導體外方法之說明書。 In another aspect, the present invention provides a kit that utilizes precursor cells (such as PPDC), and cell populations, by these cells (preferably PPDC) in various methods for eye regeneration and repair as described above The prepared conditioned medium and its components and products. When used to treat ocular degenerative conditions, or other planned treatments, the kits may include one or more cell populations or conditioned medium (including at least postpartum cells or conditioned medium derived from postpartum cells), and pharmaceutically acceptable Carrier (liquid, semi-solid or solid). The kit may optionally also include means for administering the cells and conditioned medium by injection, for example. The kit may further include instructions for using cells and conditioned medium. Prepared for field hospital use, such as military use kits may include a full-procedure supply, which includes tissue scaffolds, surgical sutures, and the like, in which cells or conditioned medium and repair of acute injuries In conjunction with. The kit used for the assay and in vitro methods as described herein may contain, for example, one or more of the following: (1) PPDC or its components, or conditioned medium or other products of PPDC; (2) for practice in vitro Reagents for the method; (3) other cells or cell populations selected as appropriate; and (4) instructions for in vitro methods.
在另一態樣中,本發明亦提供用於存庫(banking)本發明之組織、細胞、細胞群、條件培養基、及細胞組分。如上文所討論,細胞及條件培養基易於凍存。因此本發明提供凍存細胞於存庫中之方法,其中該些細胞經冷凍儲存且與基於細胞之免疫、生化及基因性質之細胞的完整表徵相關聯。冷凍細胞可經解凍及擴增或直 接用於自體、同基因、或同種異體治療,其取決於程序之要求及患者之需要。較佳地,關於每一凍存樣本之資訊儲存於電腦中,其可基於外科醫師、程序及患者之要求來搜尋,其中合適的匹配係基於細胞或族群之表徵來得到。較佳地,使本發明之細胞生長且擴增至所要量之細胞,且治療性細胞組成物係在基質或支持物存在或不存在下分開製備或以共培養之形式製備。儘管對於一些應用而言,使用新鮮製備之細胞可為較佳的,但是可藉由將細胞冷凍且將資訊輸入於電腦中以將電腦條目與樣本相關聯來將剩餘物凍存且存庫。即使在不需要將來源或捐贈者與此類細胞之接受者匹配的情況中,出於免疫目的,存庫系統使其易於將例如存庫細胞之所需生化或基因性質與治療需要匹配。在找到與所需性質相匹配之存庫樣本後,可擷取樣本且製備以供治療使用。如本文所述所製備之細胞溶解產物、ECM或細胞組分亦可經凍存或以其他方式保留(例如,藉由凍乾)且根據本發明存庫。 In another aspect, the invention also provides tissues, cells, cell populations, conditioned medium, and cell components for banking the invention. As discussed above, cells and conditioned medium are easy to freeze. Therefore, the present invention provides a method for cryopreserving cells in storage, wherein the cells are stored frozen and associated with the complete characterization of cells based on the immune, biochemical, and genetic properties of the cells. Frozen cells can be thawed and expanded or directly used for autologous, allogenic, or allogeneic treatment, depending on the requirements of the procedure and the needs of the patient. Preferably, the information about each frozen sample is stored in a computer, which can be searched based on the requirements of the surgeon, the procedure, and the patient, where the appropriate match is obtained based on the characterization of the cell or population. Preferably, the cells of the present invention are grown and expanded to the desired amount of cells, and the therapeutic cell composition is prepared separately or in the form of co-culture in the presence or absence of a matrix or support. Although for some applications, it may be preferable to use freshly prepared cells, the remaining material can be frozen and stored by freezing the cells and entering the information in a computer to associate the computer entry with the sample. Even in cases where there is no need to match the source or donor to the recipient of such cells, the storage system makes it easy to match, for example, the desired biochemical or genetic properties of the stored cells with the treatment needs. After finding a stock sample that matches the desired properties, a sample can be taken and prepared for treatment. Cell lysates, ECM or cell components prepared as described herein can also be frozen or otherwise retained (eg, by lyophilization) and stored in accordance with the present invention.
提供以下實例以更詳細描述本發明。該等實例意欲說明而非限制本發明。 The following examples are provided to describe the invention in more detail. These examples are intended to illustrate rather than limit the invention.
以下縮寫可出現於實例和說明書之其他地方及申請專利範圍中:ANG2(或Ang2)為血管生成素2;APC為抗原呈現細胞;BDNF為腦衍生神經營養因子(brain-derived neurotrophic factor);bFGF為鹼性纖維母細胞生長因子(basic fibroblast growth factor);bid(BID)為「一日兩次」(每天兩次);CK18為細胞角蛋白(cytokeratin)18;CNS為中樞神經系統;CXC配體3為趨化激素受 體配體(chemokine receptor ligand)3;DMEM為達爾伯克(Dulbecco's)最低必需培養基;DMEM:lg(或DMEM:Lg、DMEM:LG)為具有低葡萄糖之DMEM;EDTA為乙二胺四乙酸;EGF(或E)為表皮生長因子(epidermal growth factor);FACS為螢光活化細胞分選(fluorescent activated cell sorting);FBS為胎牛血清;FGF(或F)為纖維母細胞生長因子(fibroblast growth factor);GCP-2為顆粒性細胞趨化蛋白質(granulocyte chemotactic protein)-2;GDNF為膠細胞衍生神經營養因子(glial cell-derived neurotrophic factor);GF AP為膠細胞纖維酸性蛋白質(glial fibrillary acidic protein);HB-EGF為肝素結合表皮生長因子(heparin-binding epidermal growth factor);HCAEC為人類冠狀動脈內皮細胞(Human coronary artery endothelial cell);HGF為肝細胞生長因子(hepatocyte growth factor);hMSC為人類間葉幹細胞(Human mesenchymal stem cell);HNF-1α為肝細胞特異性轉錄因子(hepatocyte-specific transcription factor);HVVEC為人類臍靜脈內皮細胞(Human umbilical vein endothelial cell);I309為趨化激素及CCR8受體之配體;IGF-1為類胰島素生長因子(insulin-like growth factor)1;IL-6為介白素(interleukin)6;IL-8為介白素8;K19為角蛋白(keratin)19;K8為角蛋白8;KGF為角質細胞生長因子(keratinocyte growth factor);LIF為白血病抑制因子(leukemia inhibitory factor);MBP為髓鞘鹼性蛋白(myelin basic protein);MCP-1為單核細胞趨化蛋白質(monocyte chemotactic protein)1;MDC為巨噬細胞衍生趨化激素(macrophage-derived chemokine);MIP1α為巨噬細胞發炎蛋白質(macrophage inflammatory protein)1α;MIP1β為巨噬細胞發炎蛋白質1β;MMP為基質金屬蛋白酶(matrix metalloprotease,MMP);MSC為間葉幹細胞(mesenchymal stem cell);NHDF為正常人類皮膚纖維母細胞(Normal Human Dermal Fibroblast);NPE為神經前驅細胞擴增培養基(Neural Progenitor Expansion media);NT3為神經營養蛋白(neurotrophin)3;04為寡樹突細胞(oligodendrocyte)或膠細胞分化標記(glial differentiation marker)04;PBMC為周邊血液單核細胞(Peripheral blood mononuclear cell);PBS為磷酸鹽緩衝液(phosphate buffered saline);PDGF-CC為血小板衍生生長因子(platelet derived growth factor)C;PDGF-DD為血小板衍生生長因子D;PDGFbb為血小板衍生生長因子bb;PO為「經口(per os)」(經由嘴巴);PNS為周邊神經系統(peripheral nervous system);Rantes(或RANTES)為調控活化、正常T細胞表現與分泌(regulated on activation,normal T cell expressed and secreted);rhGDF-5為重組人類生長及分化因子(recombinant human growth and differentiation factor)5;SC為皮下(subcutaneously);SDF-1α為基質衍生因子(stromal-derived factor)1α;SHH為音蝟(sonic hedgehog);SOP為標準作業程序;TARC為胸腺與活化調控趨化激素(thymus and activation-regulated chemokine);TCP為組織培養塑膠(Tissue culture plastic);TCPS為組織培養聚苯乙烯;TGFβ2為轉形生長因子(transforming growth factor)β2;TGF β-3為轉形生長因子β-3;TIMP1為基質金屬蛋白酶 組織抑制因子(tissue inhibitor of matrix metalloproteinase)1;TPO為血小板生成素(thrombopoietin);TUJ1為BIII微管蛋白(tubulin);VEGF為血管內皮生長因子(vascular endothelial growth factor);vWF為馮威里氏因子(von Willebrand factor);及αFP為α-胎蛋白(fetoprotein)。 The following abbreviations may appear elsewhere in the examples and specifications and in the scope of patent applications: ANG2 (or Ang2) is Angiopoietin 2; APC is an antigen presenting cell; BDNF is brain-derived neurotrophic factor; bFGF Basic fibroblast growth factor (basic fibroblast growth factor); bid (BID) is "twice a day" (twice a day); CK18 is cytokeratin 18; CNS is the central nervous system; CXC Body 3 is chemokine receptor ligand 3; DMEM is the minimum essential medium for Dulbecco's; DMEM: lg (or DMEM: Lg, DMEM: LG) is DMEM with low glucose; EDTA Ethylenediaminetetraacetic acid; EGF (or E) is epidermal growth factor; FACS is fluorescent activated cell sorting; FBS is fetal bovine serum; FGF (or F) is fiber Fibroblast growth factor; GCP-2 is granulocyte chemotactic protein-2; GDNF is a glial cell-derived neurotrophic factor; GF AP is glial fibrillary acidic protein; HB-EGF is heparin-binding epidermal growth factor; HCAEC is human coronary artery endothelial cell; HGF is Hepatocyte growth factor; hMSC is Human mesenchymal stem cell; HNF-1α is hepatocyte-specific transcription factor; HVVEC is human umbilical vein endothelial cell (Human umbilical vein endothelial cell); I309 is a ligand for chemokine and CCR8 receptor; IGF-1 is insulin-like growth factor 1; IL-6 is interleukin 6; IL- 8 is interleukin 8; K19 is keratin 19; K8 is keratin 8; KGF is keratinocyte growth factor; LIF is leukemia inhibitory factor; MBP is myelin base Myelin basic protein; MCP-1 is a monocyte chemotactic protein 1; MDC is a macrophage-derived chemokine (ma crophage-derived chemokine); MIP1α is macrophage inflammatory protein 1α; MIP1β is macrophage inflammatory protein 1β; MMP is matrix metalloprotease (MMP); MSC is mesenchymal stem cell ); NHDF is Normal Human Dermal Fibroblast; NPE is Neural Progenitor Expansion media; NT3 is neurotrophin 3; 04 is oligodendrocyte ) Or glial differentiation marker 04; PBMC is peripheral blood mononuclear cell; PBS is phosphate buffered saline; PDGF-CC is platelet derived growth factor (platelet derived growth factor) growth factor) C; PDGF-DD is platelet-derived growth factor D; PDGFbb is platelet-derived growth factor bb; PO is "per os" (via mouth); PNS is peripheral nervous system; Rantes (Or RANTES) is to regulate activation, normal T cell performance and secretion (regulated on activat ion, normal T cell expressed and secreted); rhGDF-5 is a recombinant human growth and differentiation factor 5; SC is subcutaneously; SDF-1α is a stromal-derived factor 1α; SHH for sonic hedgehog; SOP for standard operating procedures; TARC for thymus and activation-regulated chemokine; TCP for tissue culture plastic; TCPS for tissue culture Polystyrene; TGF β2 is transforming growth factor β2; TGF β-3 is transforming growth factor β-3; TIMP1 is tissue inhibitor of matrix metalloproteinase 1; TPO is platelets Thrombopoietin; TUJ1 is BIII tubulin; VEGF is vascular endothelial growth factor; vWF is von Willebrand factor; and αFP is α-fetoprotein .
以下實例進一步說明但不限制本發明。 The following examples further illustrate but do not limit the invention.
已充分確定,來自皇家外科學院(RCS)大鼠之視網膜色素上皮(RPE)細胞由於Mertk基因之無效突變而表現出視桿細胞外節(ROS)吞噬受損。(Feng W.等人,J Biol Chem.2002,10:277(19):17016-17022)。已顯示,將人類臍組織衍生細胞(hUTC)經視網膜下注射至RCS大鼠眼睛中改良視覺銳度且似乎改善視網膜變性。(US 2010/0272803)。在此實例中,以衍生自hUTC之條件培養基(CM)處理,使體外失養性RPE細胞中恢復對ROS之吞噬。 It has been well established that retinal pigment epithelium (RPE) cells from the Royal College of Surgeons (RCS) rats exhibit impaired phagocytic extracellular section (ROS) phagocytosis due to invalid mutations in the Mertk gene. (Feng W. et al., J Biol Chem. 2002, 10: 277 (19): 17016-17022). It has been shown that human umbilical tissue-derived cells (hUTC) are injected subretinally into the eyes of RCS rats to improve visual acuity and appear to improve retinal degeneration. (US 2010/0272803). In this example, treatment with conditioned medium (CM) derived from hUTC restores phagocytosis of ROS in in vitro dystrophic RPE cells.
檢測hUTC條件培養基以:1)評估使用hUTC CM新製劑對失養性RPE吞噬之影響;2)將可接受品質之RNA自經CM處理及未處理失養性RPE分離以用於藉由RNA-Seq之基因表現分析;3)檢測選定RTK配體是否可增加無法使用Mertk傳訊路徑之 RCS大鼠的RPE吞噬水準;及4)測試活化非RTK之不同受體之其他非RTK配體是否可展現出類似功能。 The hUTC conditioned medium was tested to: 1) evaluate the effect of using the new hUTC CM preparation on phagocytosis of deferent RPE; 2) separate RNA of acceptable quality from CM-treated and untreated deferent RPE for use by RNA-Seq Gene expression analysis; 3) Test whether the selected RTK ligand can increase the RPE phagocytosis level of RCS rats that cannot use the Mertk signaling pathway; and 4) Test whether other non-RTK ligands that activate different receptors other than RTK can show similar Features.
人類臍組織衍生細胞(hUTC)係獲自以下實例14至26中所述、及詳細描述於美國專利第7,524,489號和第7,510,873號、及美國公開申請案第2005/0058634號中之方法,各以引用方式併入本文中。簡言之,捐贈者同意活產後捐贈之人類臍帶係得自國家疾病研究交換中心(National Disease Research Interchange,Philadelphia,PA)。將組織絞碎且用酶消化。在用含有50U/mL膠原蛋白酶(Sigma,St.Louis)之混合物的達爾伯克改良伊格爾培養基(DMEM)-低葡萄糖(Lg)(Invitrogen,Carlsbad,CA)培養基幾乎完全消化之後,使細胞懸浮液過濾通過70[tm過濾器,且將上清液以350g離心。將分離之細胞於DMEM-Lg中清洗幾次,且將其以5,000個細胞/cm2之密度接種於含有15%(v/v)FBS(Hyclone,Logan,Utah)及4mM L-麩醯胺酸(Gibco,Grand Island,NY)之DMEM-Lg培養基中。當細胞達到大致70%長滿時,使用TrypLE(Gibco,Grand Island,NY)繼代。在數次繼代之後收集細胞並存庫。 Human umbilical tissue-derived cells (hUTC) are obtained from the methods described in Examples 14 to 26 below and described in detail in US Patent Nos. 7,524,489 and 7,510,873, and US Published Application No. 2005/0058634 The way of quotation is incorporated herein. In short, the donor agreed that the human umbilical cord donated after live birth was obtained from the National Disease Research Interchange (Philadelphia, PA). The tissue was minced and digested with enzymes. After almost complete digestion with Dulbecco's modified Eagle's medium (DMEM) -low glucose (Lg) (Invitrogen, Carlsbad, CA) medium containing a mixture of 50 U / mL collagenase (Sigma, St. Louis), the cells were allowed to The suspension was filtered through a 70 [tm filter, and the supernatant was centrifuged at 350 g. The separated cells were washed several times in DMEM-Lg, and they were seeded at a density of 5,000 cells / cm 2 in 15% (v / v) FBS (Hyclone, Logan, Utah) and 4 mM L-glutamine Acid (Gibco, Grand Island, NY) in DMEM-Lg medium. When the cells reached approximately 70% overgrowth, trypLE (Gibco, Grand Island, NY) was used for subculture. Cells were collected and pooled after several passages.
RPE細胞之初代培養:RPE細胞係獲自6至11日齡之色素正常(RCS rdy+/p+)(同源異基因對照)或失養性(RCS rdy-/p+)大鼠。角膜緣之前的眼睛前部係經移除。小心移除視網膜且將眼杯於4%(w/v)分散酶(0.8U/mg,Roche Diagnostics,Mannheim, Germany)中培養20至30分鐘。將RPE片移除、懸浮於生長培養基(DMEM+10% FBS[新紙20%]+Pen(200U/ml)/Strep(200μg/ml))中、以胰蛋白酶處理研製、且接種於8孔腔室玻片孔中或接種於放置於24孔盤之孔中的圓形玻璃蓋玻片上。將細胞在37℃下於5% v/v CO2中培養。 Cultured RPE cells beginning: RPE cell lines were obtained from 6-11 days of age normal dye (RCS rdy + / p +) ( homologous allogeneic control) or sexual dystrophy (RCS rdy- / p +) rats. The front of the eye before the limbus is removed. Carefully remove the retina and place the eye cup at 4% (w / v) dispase ( 0.8 U / mg, Roche Diagnostics, Mannheim, Germany) for 20 to 30 minutes. The RPE sheet was removed, suspended in growth medium (DMEM + 10% FBS [new paper 20%] + Pen (200U / ml) / Strep (200 μg / ml)), developed by trypsin treatment, and inoculated in 8 wells Chamber slide holes or inoculate on round glass coverslips placed in the wells of a 24-well plate. The cells were cultured in 5% v / v CO 2 at 37 ° C.
RPE細胞之磺醯羅丹明(sulforhodamine)染色:將RPE培養物維持在含有磺醯羅丹明(40μg/ml最終濃度)之生長培養基中24h至72h。在添加ROS之前將細胞染色36h至48h。在添加ROS之前6h至18h,將含磺醯羅丹明之培養基移除,且將培養物維持於更換數次之新鮮生長培養基中。 RPE cell sulforhodamine (sulforhodamine) staining: maintain RPE culture in sulforhodamine (40 μg / ml final concentration) growth medium containing 24h to 72h. Cells were stained for 36h to 48h before adding ROS. Between 6h and 18h before the addition of ROS, the sulforhodamine-containing medium was removed, and the culture was maintained in fresh growth medium that was replaced several times.
大鼠光受體OS之分離:眼睛在光起始之後數小時係獲自2至4週齡或6至8週齡的Long Evans大鼠。將視網膜分離,用Polytron(8mm均質機)或Dounce玻璃均質機均質化,分層於27%至50%線性蔗糖梯度頂部,並在4℃下於SW41轉子(240,000xg)中以38,000rpm離心1小時。收集頂部兩個ROS帶,將其以HBSS稀釋,並於HB-4轉子(8000xg)中以7000rpm離心10分鐘以使ROS成為團塊。 Isolation of rat photoreceptor OS : Eyes were obtained from Long Evans rats 2 to 4 weeks old or 6 to 8 weeks old several hours after the initiation of light. The retina was separated, homogenized with a Polytron (8mm homogenizer) or Dounce glass homogenizer, layered on top of a 27% to 50% linear sucrose gradient, and centrifuged at 38,000rpm in a SW41 rotor (240,000xg) at 4 ° C hour. The top two ROS bands were collected, diluted with HBSS, and centrifuged at 7000 rpm for 10 minutes in an HB-4 rotor (8000xg) to make ROS clumps.
ROS之FITC染色:將ROS團塊以每團塊約1ml再懸浮於無血清培養基(僅MEM基礎培養基)中。添加FITC儲備液(2mg/ml於0.1M碳酸氫鈉中,pH 9.0至9.5)至最終濃度10μg/ml,且在室溫下培養1h。將經FITC染色之ROS藉由於微量離心機中以 8000rpm離心10分鐘以成為團塊,再懸浮於生長培養基(MEM20),計數,且稀釋成107/ml之最終濃度。 FITC staining of ROS: the ROS pellet of about 1ml per pellet was resuspended in serum-free medium (MEM basal medium only) on. FITC stock solution (2 mg / ml in 0.1 M sodium bicarbonate, pH 9.0 to 9.5) was added to a final concentration of 10 μg / ml, and incubated at room temperature for 1 h. FITC-stained ROS were pelleted by centrifugation at 8000 rpm for 10 minutes in a microcentrifuge, resuspended in growth medium (MEM20), counted, and diluted to a final concentration of 107 / ml.
hUTC條件培養基(CM):製備三組hUTC條件培養基(CM)及對照培養基並將其用於吞噬檢定中測試。 hUTC conditioned medium (CM) : Three sets of hUTC conditioned medium (CM) and control medium were prepared and used in phagocytosis testing.
在第1天,將hUTC以5,000個存活細胞/cm2接種於T75細胞培養瓶中之hUTC生長培養基(DMEM低葡萄糖+15% FBS+4mM L-麩醯胺酸)中。將細胞於37℃下、5% CO2培養器中培養24小時。在第2天,吸出培養基、用DPBS清洗兩次、且補充21mL之DMEM/F12完全培養基(DMEM:F12培養基+10% FBS+Pen(50U/ml)/Strep(50μg/ml))。再培養細胞54小時。亦將單獨對照培養基(DMEM:F12培養基+10% FBS+Pen(50U/ml)/Strep(50μg/ml))培養54h。在第4天,收集細胞培養物上清液及對照培養基,並將其在RT下以250g離心5min,以3mL/管等分於凍存管中,且立即冷凍於-70℃冰箱。 On day 1, hUTC was seeded at 5,000 viable cells / cm 2 in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamic acid) in T75 cell culture flasks. The cells were cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. On the second day, the medium was aspirated, washed twice with DPBS, and supplemented with 21 mL of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)). The cells were incubated for an additional 54 hours. A separate control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)) was also cultured for 54h. On the 4th day, the cell culture supernatant and control medium were collected and centrifuged at 250 g for 5 min at RT, aliquoted in 3 mL / tube into cryopreservation tubes, and immediately frozen in a -70 ° C refrigerator.
在第1天,將hUTC以5,000個存活細胞/cm2接種於T75細胞培養瓶中之hUTC生長培養基(DMEM低葡萄糖+15% FBS+4mM L-麩醯胺酸)中。將細胞於37℃下、5% CO2培養器中培養24小時。在第2天,吸出培養基、用DPBS清洗兩次、且補充21mL 之DMEM/F12無血清培養基(DMEM:F12培養基+Pen(50U/ml)/Strep(50μg/ml))。再將細胞培養54h。亦將單獨無血清對照培養基(DMEM:F12培養基+Pen(50U/ml)/Strep(50μg/ml))培養54h。在第4天,收集細胞培養物上清液及對照培養基,並將其在RT下以250g離心5min,以3mL/管等分於凍存管中,且立即冷凍於-70℃冰箱。 On day 1, hUTC was seeded at 5,000 viable cells / cm 2 in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamic acid) in T75 cell culture flasks. The cells were cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. On Day 2, the medium was aspirated, washed twice with DPBS, and supplemented with 21 mL of DMEM / F12 serum-free medium (DMEM: F12 medium + Pen (50U / ml) / Strep (50 μg / ml)). The cells were cultured for 54h. A serum-free control medium (DMEM: F12 medium + Pen (50U / ml) / Strep (50 μg / ml)) was also cultured for 54 h. On the 4th day, the cell culture supernatant and control medium were collected and centrifuged at 250 g for 5 min at RT, aliquoted in 3 mL / tube into cryopreservation tubes, and immediately frozen in a -70 ° C refrigerator.
與具有血清之CM1製劑相同且hUTC以5,000個存活細胞/cm2接種之製劑,除了細胞培養瓶為每瓶具有63mL培養基之T225瓶,且在更換培養基之後培養時間為48小時。 The preparation was the same as the CM1 preparation with serum and hUTC was inoculated at 5,000 viable cells / cm 2 , except that the cell culture flask was a T225 flask with 63 mL of culture medium per flask, and the culture time after the medium replacement was 48 hours.
與CM2相同之製劑,除了hUTC接種密度增加至10,000個存活細胞/cm2,且在更換培養基之後培養時間為48小時。 The same formulation as CM2, except that the hUTC seeding density was increased to 10,000 viable cells / cm 2 and the culture time after the medium was changed was 48 hours.
吞噬檢定:在檢定之前,將5×104個經磺醯羅丹明染色之RPE細胞接種於多孔板中,維持於MEM+20%(v/v)FBS中6天,隨後維持於MEM+5%(v/v)FBS中24h(每樣本有2或更多個重複)。在添加ROS之前3h添加新鮮培養基,且檢定係藉由以FITC-ROS(107/ml於MEM+20%(v/v)FBS中)覆蓋培養物且在37℃下培養3至19h(通常8h)。在培養結束時,將細胞劇烈清洗 以移除未結合之ROS且以2%(w/v)多聚甲醛(Sigma,St.Louis,MO)固定。 Phagocytosis test: Prior to the test, 5 × 10 4 RPE cells stained with sulforhodamine were seeded in multi-well plates, maintained in MEM + 20% (v / v) FBS for 6 days, and then maintained in MEM + 5 % (v / v) 24h in FBS (2 or more repeats per sample). Fresh medium was added 3h before the addition of ROS, and the assay was performed by covering the culture with FITC-ROS (10 7 / ml in MEM + 20% (v / v) FBS) and incubating at 37 ° C for 3 to 19h (usually 8h). At the end of the culture, the cells were vigorously washed to remove unbound ROS and fixed with 2% (w / v) paraformaldehyde (Sigma, St. Louis, MO).
RPE吞噬ROS在以下規程方面係經最佳化:初代RPE之製備及培養、ROS之製備、及吞噬檢定本身。 RPE phagocytosis of ROS is optimized in terms of the following procedures: preparation and cultivation of primary RPE, preparation of ROS, and the phagocytosis test itself.
RTK配體:所使用之RTK配體為:重組人類蝶素B2(目錄號pro-937,批號1112PEFNB2,ProSpec-Tany TechnoGene Ltd.,Israel)、重組人類BDNF(目錄號248-BD-025/CF,批號NG4012051,R&D Systems,Inc.,Minneapolis,MN)、重組人類HB-EGF(目錄號259-HE-050/CF,批號JI3012021,R&D Systems,Inc.,Minneapolis,MN)、重組人類HGF(目錄號PHG0254,批號73197181A,Life Technologies,Carlsbad,CA)、重組人類蝶素A4(目錄號E199,批號1112R245,Leinco Technologies,Inc.,St.Louis,MO)、及重組人類PDGF-DD(目錄號1159-SB-025/CF,批號OTH0412071,R&D Systems,Inc.,Minneapolis,MN)。個別RTK配體儲備液之重構係根據供應商之資料表:重組人類BDNF及HB-EGF分別以100μg/mL及250μg/mL重構於無菌PBS中。重組人類HGF以500μg/mL重構於無菌蒸餾水中。重組人類蝶素A4以100μg/mL重構於無菌PBS中。重組人類PDGF-DD以100μg/mL重構於無菌4mM HCl中。經重構之儲備液經等分且冷凍於-70℃冰箱。 RTK ligands: The RTK ligands used were: recombinant human butterfly B2 (catalogue number pro-937, batch number 1112PEFNB2, ProSpec-Tany TechnoGene Ltd., Israel), recombinant human BDNF (catalogue number 248-BD-025 / CF , Batch number NG4012051, R & D Systems, Inc., Minneapolis, MN), recombinant human HB-EGF (catalogue number 259-HE-050 / CF, batch number JI3012021, R & D Systems, Inc., Minneapolis, MN), recombinant human HGF (catalog PHG0254, batch number 73197181A, Life Technologies, Carlsbad, CA), recombinant human butterfly A4 (catalog number E199, batch number 1112R245, Leinco Technologies, Inc., St. Louis, MO), and recombinant human PDGF-DD (catalog number 1159 -SB-025 / CF, lot number OTH0412071, R & D Systems, Inc., Minneapolis, MN). The reconstitution of individual RTK ligand stock solutions was based on the supplier's data sheet: recombinant human BDNF and HB-EGF were reconstituted in sterile PBS at 100 μg / mL and 250 μg / mL, respectively. Recombinant human HGF was reconstituted in sterile distilled water at 500 μg / mL. Recombinant human butterfly A4 was reconstituted in sterile PBS at 100 μg / mL. Recombinant human PDGF-DD was reconstituted in sterile 4mM HCl at 100μg / mL. The reconstituted stock solution was aliquoted and frozen in the refrigerator at -70 ° C.
將培養基更換成MEM+5%(v/v)FBS(MEM5),且將配體以200ng/ml添加至失養性細胞,培養24h,接著在配體存在 下添加ROS,且使細胞進行吞噬檢定。正常RPE重複係預培養於MEM5中且進行吞噬檢定作為對照。 The medium was changed to MEM + 5% (v / v) FBS (MEM5), and the ligand was added to the dystrophic cells at 200ng / ml, cultured for 24h, then ROS was added in the presence of the ligand, and the cells were subjected to phagocytosis assay . Normal RPE repeats were pre-cultured in MEM5 and phagocytosis assay was used as a control.
非RTK配體:所使用之非RTK配體為:重組人類玻璃連接蛋白(目錄號2308-VN-050,批號NBH0713021,R&D Systems,Inc.,Minneapolis,MN)、重組人類TGF-β1(目錄號240-B-010/CF,批號AV5412113,R&D Systems,Inc.,Minneapolis,MN)、重組人類IL-6(目錄號206-IL-010/CF,批號OJZ0712041,R&D Systems,Inc.,Minneapolis,MN)、及人類內皮素-1(目錄號hor-307,批號1211PEDN112,ProSpec-Tany TechnoGene Ltd.,Israel)。個別非RTK配體儲備液之重構係根據供應商之資料表:重組人類玻璃連接蛋白及IL-6分別以100μg/mL重構於無菌PBS中。重組人類TGF-β1以100μg/mL重構於無菌4mM HCl中。重組人類內皮素-1以100μg/mL重構於無菌18MΩ-cm H2O中。經重構之儲備液經等分且冷凍於-70℃冰箱。 Non- RTK ligands: The non-RTK ligands used are: recombinant human vitreous connexin (catalog number 2308-VN-050, batch number NBH0713021, R & D Systems, Inc., Minneapolis, MN), recombinant human TGF-β1 (catalog number 240-B-010 / CF, batch number AV5412113, R & D Systems, Inc., Minneapolis, MN), recombinant human IL-6 (catalog number 206-IL-010 / CF, batch number OJZ0712041, R & D Systems, Inc., Minneapolis, MN ), And human endothelin-1 (catalog number hor-307, batch number 1211PEDN112, ProSpec-Tany TechnoGene Ltd., Israel). The reconstitution of individual non-RTK ligand stock solutions was reconstituted in sterile PBS at 100 μg / mL of recombinant human vitronectin and IL-6 according to the supplier's data sheet. Recombinant human TGF-β1 was reconstituted in sterile 4mM HCl at 100μg / mL. Recombinant human endothelin-1 was reconstituted in sterile 18 MΩ-cm H 2 O at 100 μg / mL. The reconstituted stock solution was aliquoted and frozen in the refrigerator at -70 ° C.
條件培養基之吞噬救援活性檢定:失養性(D)RPE之重複經各1ml之條件培養基培養約24h。至於對照組,以相同時間將同源異基因對照(N)RPE之重複培養於對照培養基(DMEM:F12培養基+10% FBS+Pen(50U/ml)/Strep(50μg/ml))中。在培養之後,將條件培養基及對照培養基移除,以新鮮MEM5替換,且使RPE進行吞噬檢定。 The phagocytosis rescue activity test of conditioned medium: the repeat of dystrophic (D) RPE was cultured for about 24 hours in 1 ml of conditioned medium each. As for the control group, the homologous allogeneic control (N) RPE was repeatedly cultured in the control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)) at the same time. After the cultivation, the conditioned medium and the control medium were removed, replaced with fresh MEM5, and RPE was subjected to phagocytosis assay.
檢測非RTK配體對RCS RPE細胞吞噬之作用的檢定:hUTC CM3係用作檢定之陽性對照。在內皮素-1、TGF-β1、及 IL-6的測試方面,失養性(D)RPE經各1ml之條件培養基培養24h。然後,將條件培養基移除,以新鮮MEM+5%(v/v)FBS(MEM5)替換,且進行吞噬檢定(於MEM5中饋以ROS 8h)。至於其他對照,將正常與失養性RPE培養於MEM5中24h且進行吞噬檢定。失養性RPE細胞係經重組人類內皮素-1、TGF-β1或IL-6以200ng/mL培養於MEM5中24h,隨後添加ROS於含有重組人類內皮素-1、TGF-β1或IL-6之MEM5中(添加ROS時不更換培養基)以進行吞噬檢定。 Assay for detecting the effect of non- RTK ligands on phagocytosis of RCS RPE cells: hUTC CM3 is used as a positive control for the assay. In the test of endothelin-1, TGF-β1, and IL-6, the dystrophic (D) RPE was cultured in 1 ml of conditioned medium for 24 hours. Then, the conditioned medium was removed, replaced with fresh MEM + 5% (v / v) FBS (MEM5), and a phagocytosis test was performed (feed ROS 8h in MEM5). As for other controls, normal and deferent RPE were cultured in MEM5 for 24h and subjected to phagocytosis assay. The dystrophic RPE cell line was cultured in MEM5 at 200 ng / mL with recombinant human endothelin-1, TGF-β1 or IL-6 for 24 h, and then added ROS to cells containing recombinant human endothelin-1, TGF-β1 or IL-6 Perform phagocytosis assay in MEM5 (do not change medium when adding ROS).
在玻璃連接蛋白之測試方面,ROS係在37℃下以對照培養基(DMEM+10% FBS)或條件培養基於CO2細胞培養器中預培養24h。同時,ROS係於MEM+20%(v/v)FBS(MEM20)中以各種濃度之人類重組玻璃連接蛋白(4、2、1、0.5μg/ml)分別在37℃下於CO2細胞培養器中預培養24h。在培養之後,將ROS離心沉降而不清洗,再懸浮於MEM20中,且在MEMS存在下饋至失養性RPE細胞以進行吞噬檢定。至於對照組,將單獨正常RPE或單獨失養性RPE培養於MEM20中,隨後在未處理ROS(再懸浮於MEM20中且饋至RPE細胞)存在下更換成MEM5以進行吞噬檢定。 For the test of vitronectin, ROS was pre-cultured in CO 2 cell incubator with control medium (DMEM + 10% FBS) or conditioned medium at 37 ° C for 24h. At the same time, ROS were cultured in MEM + 20% (v / v) FBS (MEM20) with various concentrations of human recombinant glass connexin (4, 2, 1, 0.5μg / ml) at 37 ℃ in CO 2 cells Pre-incubated in the instrument for 24h. After culturing, the ROS was centrifuged without washing, resuspended in MEM20, and fed to the dystrophic RPE cells in the presence of MEMS for phagocytosis assay. As for the control group, normal RPE alone or deprived RPE alone was cultured in MEM20, and then replaced with MEM5 in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) for phagocytosis assay.
成像及定量:藉由相位差及螢光顯微鏡法,使用裝備有落射螢光光學、螢光顯微鏡、及數位照相機之倒立顯微鏡檢測活RPE細胞。結合至細胞表面之FITC-ROS、經攝入之FITC-ROS、及吞噬溶酶體之識別係如McLaren等人(Invest Ophthalmol Vis Sci.,1993;34(2):317-326.)中所定義。經結合及攝入之ROS之定量係於蓋 玻片上之固定細胞上進行。以250x放大率用適當的過濾器及柵格(視野大小(field size),40×40μm)進行計數。計數代表性視野(每培養物10至15個視野)中之各種類型的細胞,且將數據表達為來自2至3個分開實驗之每個時間點所獲得之彙集值的平均值。藉由司徒頓t檢定(Student's t-test)檢驗成對數據之統計顯著性,且統計顯著性被定於p<0.05。 Imaging and quantification: By phase contrast and fluorescence microscopy, live RPE cells are detected using an inverted microscope equipped with epi-fluorescence optics, fluorescence microscope, and digital camera. The recognition of FITC-ROS bound to the cell surface, FITC-ROS ingested, and phagolysosome is as described in McLaren et al . (Investor Ophthalmol Vis Sci., 1993; 34 (2): 317-326.) definition. Quantification of bound and ingested ROS is performed on fixed cells on the coverslip. Count at 250x magnification with an appropriate filter and grid (field size, 40 × 40 μm). Cells of various types in representative fields (10 to 15 fields per culture) were counted, and the data was expressed as the average of pooled values obtained from each time point of 2 to 3 separate experiments. The statistical significance of the paired data was tested by Student's t-test, and the statistical significance was determined at p <0.05.
檢定接受標準:吞噬之絕對水準在實驗中取決於多種因素而有所不同,包括經分離之RPE及經製備之ROS的品質。特別使用相同譜系(亦即,相同收集及製備之時間)之RPE,以比較不同處理對細胞之作用。若正常吞噬水準相較於失養性吞噬水準之關係為大致1:0.3,則判斷檢定為合理的。 Acceptance Criteria for Testing: The absolute level of phagocytosis varies in the experiment depending on various factors, including the quality of the isolated RPE and the prepared ROS. In particular, RPEs of the same lineage (ie, the same collection and preparation time) are used to compare the effects of different treatments on cells. If the relationship between the normal phagocytosis level and the dystrophy phagocytosis level is approximately 1: 0.3, the test is judged to be reasonable.
相對吞噬:相對吞噬為失養性RPE吞噬水準相較於作為參考點之同源異基因對照(正常)吞噬水準所顯示者。吞噬水準可被表達為ROS平均數/視野、或ROS平均數/細胞。 Relative phagocytosis: Relative phagocytosis is the level of phagocytic RPE phagocytosis compared to the reference (normal) allogeneic control (normal) phagocytosis level. The level of phagocytosis can be expressed as mean ROS / field of view, or mean ROS / cell.
RNA之分離。將hUTC以5,000個細胞/cm2接種且於含有15%(v/v)FBS(Hyclone,Logan,Utah)及4mM L-麩醯胺酸(Gibco,Grand Island,NY)之DMEM-Lg培養基中生長24小時,接著將培養基更換成含有10%(v/v)FBS之DMEM:F12培養基且再生長48小時。隨後收集細胞以用於總RNA萃取及DNA移除,使用Qiagen RNAeasy萃取及柱上DNAse套組(Qiagen,Valencia,CA)。使用NanoDrop 1000分光光度計(Thermo Fisher Scientific,'Waltham,MA)及Agilent 2100生物分析儀(Agilent Technologies,Santa Clara,CA) 測定樣本中RNA之完整性及量。由Expression Analysis Inc.,Durham,NC進行文庫製備及定序。根據製造商之說明書使用Illumina之TruSeq RNA-Seq樣本製備套組製備RNA庫,且以Illumina之HiSeq 2000定序。使用軟體ArrayStudio 6.1版將定序讀數對映於參考人類基因體(GRCh37修補(patch)8)。使用每百萬對映讀數每千鹼基轉錄本之片段(FPKM)計算基因表現。 RNA isolation. Inoculate hUTC at 5,000 cells / cm 2 in DMEM-Lg medium containing 15% (v / v) FBS (Hyclone, Logan, Utah) and 4 mM L-glutamic acid (Gibco, Grand Island, NY) After growing for 24 hours, the medium was then replaced with DMEM: F12 medium containing 10% (v / v) FBS and regenerated for 48 hours. Cells were then collected for total RNA extraction and DNA removal, using Qiagen RNAeasy extraction and on-column DNAse kits (Qiagen, Valencia, CA). A NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, 'Waltham, MA) and an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) were used to determine the integrity and amount of RNA in the sample. Library preparation and sequencing were performed by Expression Analysis Inc., Durham, NC. The RNA library was prepared using Illumina's TruSeq RNA-Seq sample preparation kit according to the manufacturer's instructions, and sequenced with Illumina's HiSeq 2000. The software ArrayStudio version 6.1 was used to map the sequenced readings to the reference human genome (GRCh37 patch 8). Gene performance was calculated using fragments per kilobase transcript (FPKM) per million enantiomeric readings.
在分離之後,首先將RCS RPE細胞以三重複接種於96孔板中約一週,將培養基更換成hUTC條件培養基或對照培養基之時間點視為0h。根據製造商的規程,使用Trizol(Life Technologies,Carlsbad,CA),分別在2、4、8、及24h時萃取RNA。將各時間點自三重複萃取之RNA彙集為一個樣本。樣本之濃度與260/280吸光度比係以分光光度法測定。 After separation, the RCS RPE cells were first seeded in 96-well plates in triplicate for about one week, and the time point when the medium was replaced with hUTC conditioned medium or control medium was regarded as 0h. According to the manufacturer's protocol, Trizol (Life Technologies, Carlsbad, CA) was used to extract RNA at 2, 4, 8, and 24 h, respectively. The RNA extracted from the triplicate at each time point was pooled into one sample. The ratio of the sample concentration to the 260/280 absorbance is determined spectrophotometrically.
失養性RPE之重複(各約2.4×104)係經條件培養基處理或不處理,如以下方案所示: The duplication of defertile RPE (about 2.4 × 10 4 each ) was treated with conditioned medium or not, as shown in the following scheme:
如方法中所述,測試三種條件培養基製劑(CM1、CM2、CM3)對失養性RPE的吞噬救援活性及與正常RPE之比較。 As described in the method, three conditioned medium preparations (CM1, CM2, CM3) were tested for phagocytic rescue activity on deferent RPE and compared with normal RPE.
CM1:CM1係測試兩次(圖1A及圖1B),一次使用褐色正常RPE,另一次使用色素RPE。觀察吞噬救援活性。應注意到,在圖1A中,色素失養性RPE之吞噬基礎水準幾乎為褐色套頭(tan hooded)正常RPE之水準的50%,超過接受範圍(<正常吞噬水準之30%)。亦測試無血清之CM1培養基之作用(圖2)。具有及不具有無血清之CM1的失養性細胞之間的差異具統計顯著性。 CM1: The CM1 series was tested twice (Figure 1A and Figure 1B), once using brown normal RPE and once using pigment RPE. Observe the phagocytosis rescue activity. It should be noted that in FIG. 1A, the basal level of phagocytosis of RPE is almost 50% of the normal RPE level of tan hooded, which exceeds the acceptance range (<30% of normal phagocytosis level). The effect of serum-free CM1 medium was also tested (Figure 2). The difference between the dystrophic cells with and without serum-free CM1 is statistically significant.
CM2:CM2經測試並發現缺乏活性(圖3)。 CM2: CM2 was tested and found to lack activity (Figure 3).
CM3:在第一天更換培養基之後,CM2(不具活性)的培養時間為48h,而CM1(具活性)的培養時間為54h。為了獲得活性條件培養基,初始細胞接種密度及更換培養基後之細胞培養時間為應考量的兩個態樣。相較於CM2,CM3係藉由將細胞接種密度加倍並維持更換培養基後之相同培養時間來製備。將CN3檢定多次以確認活性之存在(圖4A、圖4B、圖4C),顯示高達100%之吞噬救援活性。 CM3: After the medium was changed on the first day, the cultivation time of CM2 (inactive) was 48h, and the cultivation time of CM1 (active) was 54h. In order to obtain an active conditioned medium, the initial cell seeding density and the cell culture time after changing the medium are two aspects that should be considered. Compared to CM2, CM3 is prepared by doubling the cell seeding density and maintaining the same incubation time after changing the medium. CN3 was tested multiple times to confirm the presence of activity (Figure 4A, Figure 4B, Figure 4C), showing up to 100% phagocytosis rescue activity.
分離自RCS大鼠眼睛之RPE細胞係經體外培養以用於吞噬檢定。將來自正常同源異基因對照大鼠眼睛之未處理RPE細胞用作對照以顯示正常吞噬水準。當RCS RPE與hUTC共培養(圖4A)或經hUTC條件培養基(CM)處理(圖4B)時,RCS RPE中之缺陷性吞噬恢復至正常RPE之水準。當RCS RPE係饋以經hUTC CM預培養之OS並在hUTC CM不存在下進行吞噬時,該等細胞之OS吞噬經救援(圖4C)。如所證明,hUTC分泌特定因子以促進RPE吞噬。 The RPE cell line isolated from RCS rat eyes was cultured in vitro for phagocytosis assay. Untreated RPE cells from normal homeologous control rat eyes were used as controls to show normal phagocytic levels. When RCS RPE was co-cultured with hUTC (Figure 4A) or treated with hUTC conditioned medium (CM) (Figure 4B), the defective phagocytosis in RCS RPE returned to normal RPE levels. When RCS RPE was fed with OS pre-cultured with hUTC CM and phagocytosed in the absence of hUTC CM, the OS phagocytosis of these cells was rescued (Figure 4C). As demonstrated, hUTC secretes specific factors to promote RPE phagocytosis.
BDNF及HB-EGF檢定:將失養性RPE二重複用BDNF及HB-EGF處理,並如方法中所述檢定其以及正常對照組之吞噬(圖5A、圖5B)。每個樣本觀察十至十二次。失養性細胞傾向 於顯示相較於正常細胞高於平常之吞噬率,但此不妨礙結果之解釋。BDNF顯示之吞噬救援活性高於CM3。 BDNF and HB-EGF test: Repeat the treatment of dystrophy RPE with BDNF and HB-EGF twice, and test their phagocytosis and normal control group as described in the method (Figure 5A, 5B). Each sample is observed ten to twelve times. The dystrophic cells tend to show a higher phagocytic rate than normal cells, but this does not hinder the interpretation of the results. BDNF showed higher phagocytosis rescue activity than CM3.
PDGF-DD、蝶素A4、及HGF檢定:每次取樣所計數之細胞數目係按每視野(圖6A、圖6C、圖6D)及每細胞(圖6B、圖6E)來表達。結果未受影響,因為每一框所計數之細胞數目為恆定的。相較於未處理之對照,PDGF-DD(圖6A)、蝶素A4(圖6C)及HGF(圖6D)皆上調失養性RPE細胞中之吞噬。PDGF-DD顯示最高救援作用(高於CM3)。 PDGF-DD, Pterin A4, and HGF assay: The number of cells counted per sampling is expressed per field (Figure 6A, 6C, 6D) and per cell (Figure 6B, 6E). The results were not affected because the number of cells counted in each box was constant. Compared to untreated controls, PDGF-DD (Figure 6A), Pterin A4 (Figure 6C), and HGF (Figure 6D) all upregulate phagocytosis in dystrophic RPE cells. PDGF-DD showed the highest rescue effect (higher than CM3).
蝶素B2檢定:蝶素B2顯示極高、高於CM3的吞噬救援活性。每視野(圖7A)及每細胞(圖7B)兩種結果係經測定。 Pterin B2 test: Pterin B2 showed extremely high phagocytosis rescue activity higher than CM3. Two results per field (Figure 7A) and per cell (Figure 7B) were determined.
內皮素-1、TGF-β1、或IL-6對RCS RPE吞噬之作用:失養性RPE細胞經內皮素-1、TGF-β1、或IL-6處理,並如材料和方法中所述檢定其以及正常對照組之吞噬(圖8A至圖8C)。每個樣本觀察十次。圖8A及圖8B顯示兩次分開之檢定。圖8B中正常及失養性RPE細胞之基礎吞噬水準低於圖8A中者,其歸因於分離自大鼠之細胞變異及ROS之不同製備;由於正常吞噬水準相較於失養性吞噬水準之關係為大致1:0.3,因此認為檢定有效。為了與圖8A中結果進行比較,圖8C中的數據係經正規化,以使得正常與失養性RPE細胞之吞噬水準與圖8A的相同。hUTC CM3提高失養性RPE 細胞之吞噬,而檢定中所測試濃度(200ng/mL)之內皮素-1、TGF-β1、或IL-6對RCS RPE吞噬無作用。 The effect of endothelin-1, TGF-β1, or IL-6 on RCS RPE phagocytosis: the dystrophic RPE cells are treated with endothelin-1, TGF-β1, or IL-6, and tested as described in the materials and methods And the phagocytosis of the normal control group (Figure 8A to Figure 8C). Each sample was observed ten times. Figures 8A and 8B show two separate tests. The basic phagocytic level of normal and deferent RPE cells in Figure 8B is lower than that in Figure 8A, which is attributed to the variation of cells isolated from rats and the different preparation of ROS; due to the relationship between normal phagocytic levels and deferent phagocytic levels It is roughly 1: 0.3, so the test is considered valid. For comparison with the results in FIG. 8A, the data in FIG. 8C is normalized so that the phagocytic levels of normal and deferent RPE cells are the same as in FIG. 8A. hUTC CM3 increased the phagocytosis of dystrophic RPE cells, and the concentration (200ng / mL) of endothelin-1, TGF-β1, or IL-6 tested in the assay had no effect on RCS RPE phagocytosis.
玻璃連接蛋白對RCS RPE吞噬之作用:將失養性RPE細胞饋以用各種濃度之玻璃連接蛋白預培養的ROS,並如材料和方法中所述檢定其以及正常對照組之吞噬(圖9)。每個樣本觀察十次。 The effect of vitronectin on RCS RPE phagocytosis: The dystrophic RPE cells were fed with ROS pre-cultured with various concentrations of glass connexin, and their phagocytosis was examined as described in the materials and methods and normal control groups (Figure 9). Each sample was observed ten times.
至於用於本研究之對照培養基及hUTC條件培養基,對照培養基含有10% FBS,對照培養基中之玻璃連接蛋白之量為約500ng/ml。由於hUTC持續地分泌玻璃連接蛋白,因此hUTC條件培養基相對於對照培養基可含有較多玻璃連接蛋白。與經hUTC CM3預處理之ROS相反,經對照培養基預處理之ROS似乎對失養性RPE吞噬無作用。所有測試濃度之玻璃連接蛋白具有與對照培養基類似之作用。此等結果與先前出版物中所報告之結果一致(Edwards等人,J Cell Physiol.1986,127:293-296;Miceli等人,Invest Ophthalmol Vis Sci.,1997;38(8):1588-1597)。玻璃連接蛋白為血清之活性組分,且引起經培養之人類捐贈者眼睛分離RPE細胞的血清刺激ROS攝取(Miceli等人,1997)。然而,在大鼠RPE細胞中,血清對正常RPE吞噬與失養性RCS RPE吞噬之作用不同。Edwards等人顯示,經培養之RCS大鼠RPE細胞與正常同源異基因對照RPE細胞在無血清培養基中吞噬可相比之少量ROS。培養基中存在20%血清大幅提高(6倍)正常RPE細胞中之吞噬,但對RCS RPE細胞則無作用(Edwards等人,J Cell Physiol.1986,127:293-296)。 As for the control medium and hUTC conditioned medium used in this study, the control medium contains 10% FBS, and the amount of glass connexin in the control medium is about 500 ng / ml. Since hUTC continuously secretes vitronectin, hUTC conditioned medium may contain more glass connexin relative to the control medium. In contrast to ROS pre-treated with hUTC CM3, ROS pre-treated with control medium appeared to have no effect on the phagocytosis of deprived RPE. All test concentrations of glass connexin have a similar effect to the control medium. These results are consistent with those reported in previous publications (Edwards et al., J Cell Physiol. 1986, 127: 293-296; Miceli et al., Invest Ophthalmol Vis Sci., 1997; 38 (8): 1588-1597 ). Vitronectin is the active component of serum and causes serum from isolated human donor RPE cells to stimulate ROS uptake (Miceli et al., 1997). However, in rat RPE cells, the effect of serum on the phagocytosis of normal RPE is different from that of deferent RCS RPE. Edwards et al. Showed that cultured RCS rat RPE cells and normal homeologous control RPE cells phagocytosed in serum-free medium can be compared with a small amount of ROS. The presence of 20% serum in the medium significantly increased (6 times) the phagocytosis in normal RPE cells, but had no effect on RCS RPE cells (Edwards et al., J Cell Physiol. 1986, 127: 293-296).
當前結果指出,玻璃連接蛋白並不涉及hUTC CM媒介之RCS RPE細胞吞噬增強。 The current results indicate that vitronectin does not involve enhanced phagocytosis of hUTC CM-mediated RCS RPE cells.
如方法中所述,進行數輪此實驗以獲得必要的RNA樣本。RNA樣本係用於由Expression Analysis,Inc.之RNA定序。在實驗1中,樣本8及9之RIN分數未達RNA定序標準。將該兩個樣本自定序清單移除。在實驗2中,樣本1之RIN分數未達RNA定序標準且係自定序清單移除。(定序結果如下)。 As described in the method, several rounds of this experiment were performed to obtain the necessary RNA samples. RNA samples are used for RNA sequencing by Expression Analysis, Inc. In Experiment 1, the RIN scores of samples 8 and 9 did not meet the RNA sequencing criteria. Remove the two samples from the sequencing list. In Experiment 2, the RIN score of Sample 1 did not meet the RNA sequencing criteria and was removed from the sequencing list. (The sequencing results are as follows).
hUTC表現RTK配體基因及橋分子基因係由基於RNA-Seq的hUTC轉錄體分析顯示。偵測15個RTK次家族之多個RTK配體之基因表現(表1-1)。各RTK次家族之RTK配體基因的表現水準係基於每百萬對映讀數每千鹼基轉錄本之片段(FPKM)之數值加以分類及繪圖(圖10;表1-1)。亦偵測包括MFG-E8、Gas6、蛋白質S、TSP-1及TSP-2之橋分子之基因表現(表1-3)。 hUTC expression RTK ligand gene and bridge molecule gene line were analyzed by RNA-Seq-based hUTC transcript analysis. The gene expression of multiple RTK ligands in 15 RTK subfamilies was detected (Table 1-1). The performance levels of RTK ligand genes of each RTK subfamily are classified and plotted based on the number of fragments per kilobase transcript (FPKM) per million enantiomeric readings (Figure 10; Table 1-1). The gene expression of bridge molecules including MFG-E8, Gas6, protein S, TSP-1 and TSP-2 was also detected (Table 1-3).
RCS RPE中對應RTK次家族及橋分子受體之基因表現顯示,RTK超家族可基於激酶域序列被分組成20個次家族(Robinson DR等人,Oncogene.2000;19(49):5548-5557)。偵測RCS RPE中20個RTK次家族中之18個的基因表現(表1-2)。在18個RTK次家族中有15個次家族對應hUTC中所表現之RTK配體基因。據報告用於橋分子結合之受體(Kevany BM等人,Physiology,2010;25(1):8-15)之基因表現亦在RCS RPE中偵測出(表1-4),包括整合素avP3、avP5、Axl、Tyro3、MerTK、及CD36。 The gene expression of the RTK subfamily and bridge molecule receptor in RCS RPE shows that the RTK superfamily can be grouped into 20 subfamilies based on the kinase domain sequence (Robinson DR et al., Oncogene. 2000; 19 (49): 5548-5557 ). The gene expression of 18 out of 20 RTK subfamilies in RCS RPE was detected (Table 1-2). Among the 18 RTK subfamilies, 15 subfamilies correspond to the RTK ligand genes expressed in hUTC. The gene expression of receptors reported to be used for bridge molecule binding (Kevany BM et al., Physiology, 2010; 25 (1): 8-15) was also detected in RCS RPE (Table 1-4), including integrins avP3, avP5, Axl, Tyro3, MerTK, and CD36.
使用RNA-Seq及資訊學數據分析之RCS RPE細胞與hUTC兩者之轉錄體概況顯示,RCS RPE細胞表現多個RTK基因,而hUTC表現多個RTK配體之基因(表2-1)。在hUTC中具有相對高的基因表現水準之七個RTK次家族之RTK配體係於hUTC條件培養基中量測,以與正常人類皮膚纖維母細胞(NHDF)及ARPE-19細胞之條件培養基比較。此等配體包括BDNF及NT3(Trk家族之配體)、HGF(Met家族之配體)、PDGF-DD及PDGF-CC(PDGF家族之配體)、蝶素B2(Eph家族之配體)、HB-EGF(ErbB家族之配體)、GDNF(Ret家族之配體)、以及聚集蛋白(Musk家族之配體)。 The transcript profiles of both RCS RPE cells and hUTC analyzed using RNA-Seq and informatics data showed that RCS RPE cells expressed multiple RTK genes, while hUTC expressed multiple RTK ligand genes (Table 2-1). The RTK formulations of seven RTK subfamilies with relatively high levels of gene expression in hUTC were measured in conditioned medium of hUTC to compare with the conditioned medium of normal human skin fibroblasts (NHDF) and ARPE-19 cells. These ligands include BDNF and NT3 (Trk family ligands), HGF (Met family ligands), PDGF-DD and PDGF-CC (PDGF family ligands), Pterin B2 (Eph family ligands) , HB-EGF (ErbB family ligand), GDNF (Ret family ligand), and Aggregate (Musk family ligand).
hUTC(批號NB12898P7製備自PDL20研究庫,第7頁)、ARPE-19細胞(繼代3)及NHDF(繼代10)係用於本研究。 hUTC (batch number NB12898P7 was prepared from the PDL20 research library, page 7), ARPE-19 cells (passage 3) and NHDF (passage 10) were used in this study.
人類BDNF ELISA套組(目錄號DBD00,批號311655,標準偵測範圍:62.5-4000pg/mL;靈敏度:20pg/mL)、人類HGF ELISA套組(目錄號DHG00,批號307319,標準偵測範圍:125-8000pg/mL;靈敏度:<40pg/mL)、人類PDGF-CC ELISA套組(目錄號DCC00,批號309376,標準偵測範圍:62.5-4000pg/mL;靈敏度:4.08pg/mL)、人類PDGF-DD ELISA套組(目錄號DDD00,批號310518,標準偵測範圍:31.3-2000pg/mL;靈敏度:1.67pg/mL)係來自R&D Systems,Inc.,Minneapolis,MN。HB-EGF ELISA套組(目錄號ab100531,批號GR135979-1,標準偵測範圍:16.4-4000pg/mL;靈敏度:<20pg/mL)及NT3 ELISA套組(目錄號ab100615,批號GR141281-1,標準偵測範圍:4.12-3000pg/mL;靈敏度:<4pg/mL)係來自abcam,Cambridge,MA。人類GDNF ELISA套組(目錄號RAB0205,批號0919130270,標準偵測範圍:2.74-2000pg/mL;靈敏度:2.74pg/mL)係來自Sigma,St.Louis,MI。人類蝶素B2 ELISA套組(目錄號MBS916324,批號R21199424,標準偵測範圍:15.6-1000pg/mL;靈敏度:15.6pg/mL)及聚集蛋白ELISA套組(目 錄號MBS454684,批號EDL201310110,標準偵測範圍:31.2-2000pg/mL;靈敏度:<13.6pg/mL)係來自MyBioSource,Inc.,San Diego,CA。 Human BDNF ELISA kit (Cat. No. DBD00, Lot No. 311655, Standard detection range: 62.5-4000pg / mL; Sensitivity: 20pg / mL), Human HGF ELISA kit (Cat. No. DHG00, Lot No. 307319, Standard detection range: 125 -8000pg / mL; sensitivity: <40pg / mL), human PDGF-CC ELISA kit (catalog number DCC00, batch number 309376, standard detection range: 62.5-4000pg / mL; sensitivity: 4.08pg / mL), human PDGF- The DD ELISA kit (catalog number DDD00, batch number 310518, standard detection range: 31.3-2000 pg / mL; sensitivity: 1.67 pg / mL) is from R & D Systems, Inc., Minneapolis, MN. HB-EGF ELISA kit (catalog number ab100531, batch number GR135979-1, standard detection range: 16.4-4000pg / mL; sensitivity: <20pg / mL) and NT3 ELISA kit (catalog number ab100615, batch number GR141281-1, standard Detection range: 4.12-3000pg / mL; sensitivity: <4pg / mL) from abcam, Cambridge, MA. The human GDNF ELISA kit (catalogue number RAB0205, batch number 0919130270, standard detection range: 2.74-2000pg / mL; sensitivity: 2.74pg / mL) is from Sigma, St. Louis, MI. Human Pterin B2 ELISA kit (catalogue number MBS916324, batch number R21199424, standard detection range: 15.6-1000pg / mL; sensitivity: 15.6pg / mL) and agrin ELISA kit (catalog number MBS454684, batch number EDL201310110, standard detection Range: 31.2-2000 pg / mL; sensitivity: <13.6 pg / mL) from MyBioSource, Inc., San Diego, CA.
hUTC、ARPE-19、及NHDF條件培養基之製備:在第1天,將hUTC、ARPE-19、及NHDF分別以10,000個存活細胞/cm2接種於T75細胞培養瓶中之15mL hUTC生長培養基(DMEM低葡萄糖+15% v/v FBS+4mM L-麩醯胺酸)中。細胞於37℃下5% CO2培養器中培養24h。在第2天,吸出培養基且補充21mL DMEM/F12完全培養基(DMEM/F12培養基+10% v/v FBS+50U/ml Pen/50μg/ml Strep)。再將細胞培養48h。亦將單獨對照培養基(DMEM/F12完全培養基)培養48h。在第4天,收集細胞培養物上清液及對照培養基,並將其在4℃下以250g離心5min,以0.5mL/管等分於凍存管中,且立即冷凍於-70℃冰箱。冷凍樣本係經解凍以供ELISA使用。 Preparation of conditioned medium for hUTC , ARPE-19 , and NHDF : On day 1, hUTC, ARPE-19, and NHDF were inoculated into 15mL hUTC growth medium (DMEM) in T75 cell culture flasks at 10,000 viable cells / cm 2 respectively Low glucose + 15% v / v FBS + 4mM L-glutamic acid). The cells were cultured in a 5% CO 2 incubator at 37 ° C for 24h. On Day 2, the medium was aspirated and supplemented with 21 mL of DMEM / F12 complete medium (DMEM / F12 medium + 10% v / v FBS + 50 U / ml Pen / 50 μg / ml Strep). The cells were cultured for 48h. The control medium alone (DMEM / F12 complete medium) was also cultured for 48h. On day 4, the cell culture supernatant and the control medium, and at 250 g at 4 deg.] C was centrifuged 5min, at 0.5mL / tube aliquots in cryotubes, and immediately frozen at -70 ℃ refrigerator . Frozen samples were thawed for ELISA.
於hUTC條件培養基中所量測之選定RTK配體水準係概述於表2-2中。 The selected RTK ligand levels measured in hUTC conditioned medium are summarized in Table 2-2.
表2-2. BDNF、NT3、HGF、PDGF-CC、PDGF-DD、GDNF、蝶素B2、HB-EGF、及聚集蛋白於hUTC條件培養基中之濃度。
於hUTC條件培養基中所量測之選定RTK配體水準亦與NHDF及ARPE-19條件培養基比較(以pg/mL/1×106個細胞正規化)。每48h每百萬個hUTC細胞分泌72.7pg/mL BDNF,相較於每48h每百萬個NHDF及ARPE-19細胞分別分泌20.4pg/mL及16.2pg/mL BDNF(圖11A)。對照培養基中BDNF之量不可偵測。(圖11G)。 The selected RTK ligand levels measured in hUTC conditioned medium were also compared with NHDF and ARPE-19 conditioned medium (normalized with pg / mL / 1 × 10 6 cells). 72.7 pg / mL BDNF was secreted per million hUTC cells every 48 h, compared to 20.4 pg / mL and 16.2 pg / mL BDNF secreted per million NHDF and ARPE-19 cells every 48 h (Figure 11A). The amount of BDNF in the control medium is not detectable. (Figure 11G).
hUTC及NHDF分泌少量NT3(圖11B)。ARPE-19條件培養基及對照培養基中NT3之量不可偵測。 hUTC and NHDF secrete a small amount of NT3 (Figure 11B). The amount of NT3 in ARPE-19 conditioned medium and control medium is undetectable.
每48h每百萬個hUTC細胞分泌23.7pg/mL HGF,相較於NHDF及ARPE-19分別分泌3.9pg/mL及0.4pg/mL NHDF(圖11C)。對照培養基中HGF之量不可偵測。(圖11G)。 Every 48h, 23.7 pg / mL HGF was secreted per million hUTC cells, compared with 3.9 pg / mL and 0.4 pg / mL NHDF, respectively, compared to NHDF and ARPE-19 (Figure 11C). The amount of HGF in the control medium is not detectable. (Figure 11G).
hUTC CM中PDGF-CC及PDGF-DD之量相較於NHDF及ARPE-19 CM中之量係顯示於圖11D及圖11E中。對照培養基中分別偵測出0.3pg/mL PDGF-CC及3.1pg/mL PDGF-DD。 The amounts of PDGF-CC and PDGF-DD in hUTC CM compared to NHDF and ARPE-19 CM are shown in FIGS. 11D and 11E. 0.3 pg / mL PDGF-CC and 3.1 pg / mL PDGF-DD were detected in the control medium, respectively.
每48h每百萬個hUTC細胞分泌9.5pg/mL GDNF,相較於NHDF分泌8.5pg/mL GDNF。每48h每百萬個ARPE-19細胞僅釋放痕量1.3pg/mL GDNF(圖11F)。對照培養基中GDNF之量不可偵測。(圖11G)。 Every 48h, every million hUTC cells secrete 9.5pg / mL GDNF, compared with 8.5pg / mL GDNF secreted by NHDF. Only trace amounts of 1.3 pg / mL GDNF were released per million ARPE-19 cells every 48h (Figure 11F). The amount of GDNF in the control medium is not detectable. (Figure 11G).
hUTC、NHDF、及ARPE-19之條件培養基中蝶素B2及HB-EGF之水準低於ELISA之偵測界限(分別為15.6pg/mL及20pg/mL)。hUTC、NHDF及ARPE-19條件培養基中之聚集蛋白水準類似於對照培養基。 The levels of pterin B2 and HB-EGF in the conditioned medium of hUTC, NHDF, and ARPE-19 were below the detection limit of ELISA (15.6 pg / mL and 20 pg / mL, respectively). The levels of aggregated protein in conditioned medium of hUTC, NHDF and ARPE-19 were similar to the control medium.
在RCS RPE吞噬檢定中,使用200ng/mL之劑量之BDNF、HGF、PDGF-DD、蝶素-B2及HB-EGF所進行之測試皆顯示救援RCS RPE細胞吞噬的陽性作用。(實例1)。然而,條件培養基中hUTC所分泌之此等配體之實際濃度低於所測試之水準。 In the RCS RPE phagocytosis test, tests performed with BDNF, HGF, PDGF-DD, pterin-B2, and HB-EGF at a dose of 200 ng / mL all showed a positive effect on rescue phagocytosis of RCS RPE cells. (Example 1). However, the actual concentration of these ligands secreted by hUTC in the conditioned medium is lower than the level tested.
RCS RPE細胞及hUTC兩者之轉錄體概況顯示,RCS RPE細胞表現辨識細胞凋亡細胞上之「吃我」信號的受體基因。(Erwig L-P,Cell Death and Differentiation 2008;15:243-250)。此等受體包括清道夫受體(SR-A、LOX-1、CD68、CD36、CD14)、整合素(αvβ3及αvβ5)、Axl及Tyro3之受體酪胺酸激酶、LRP-1/CD91、及PS受體穩定素1。此外,hUTC表現許多橋分子基因,包括TSP-1、TSP-2、界面活性蛋白質D(SP-D)、MFG-E8、Gas6、載脂蛋白H(apolipoprotein H)、及膜聯蛋白1(annexin 1)。檢測hUTC條件培養基中之橋分子分泌,且將水準與來自ARPE-19及正常人類皮膚纖維母細胞(NHDF)之該些水準比較。 The transcript profiles of both RCS RPE cells and hUTC show that RCS RPE cells express receptor genes that recognize the "eat me" signal on apoptotic cells. (Erwig L-P, Cell Death and Differentiation 2008; 15: 243-250). These receptors include scavenger receptors (SR-A, LOX-1, CD68, CD36, CD14), integrin (αvβ3 and αvβ5), Axl and Tyro3 receptor tyrosine kinase, LRP-1 / CD91, And PS receptor stabilizer 1. In addition, hUTC expresses many bridge molecule genes, including TSP-1, TSP-2, interface active protein D (SP-D), MFG-E8, Gas6, apolipoprotein H (apolipoprotein H), and annexin 1 (annexin 1). The secretion of bridge molecules in hUTC conditioned medium was detected and the levels were compared with those from ARPE-19 and normal human skin fibroblasts (NHDF).
hUTC(PDL20,主細胞庫編號25126057)、ARPE-19細胞(繼代3)(ATCC,Manassas,VA)及NHDF(繼代11)(Lonza,South Plainfield,NJ)係用於本研究。 hUTC (PDL20, main cell bank number 25126057), ARPE-19 cells (passage 3) (ATCC, Manassas, VA) and NHDF (passage 11) (Lonza, South Plainfield, NJ) were used in this study.
人類Gas6 ELISA套組(目錄號SK00098-01,批號20111218)及人類SP-D ELISA套組(目錄號SK00457-01,批號20111135)係來自Aviscera Bioscience,Santa Clara,CA。人類Gas6 ELISA套組之標準偵測範圍為62.5-8000pg/mL,靈敏度為31pg/ml。人類SP-D ELISA套組之標準偵測範圍為78-5000pg/mL,靈敏度為30pg/ml。人類MFG-E8 ELISA套組(目錄號DFGE80,批號307254,標準偵測範圍:62.5-4000pg/mL;靈敏度:4.04pg/mL)、人類TSP-1 ELISA套組(目錄號DTSP10,批號307182,標準偵測範圍:7.81-500ng/mL;靈敏度:0.355ng/mL)、人類TSP-2 ELISA套組(目錄號DTSP10,批號307266,標準偵測範圍:0.31-20ng/mL;靈敏度:0.025ng/mL)係來自R&D Systems,Inc.,Minneapolis,MN。載脂蛋白H人類ELISA套組(目錄號ab108814,批號GR126938,標準偵測範圍:0.625-40ng/mL,靈敏度:0.6ng/mL)係來自Abcam,Cambridge,MA。人類膜聯蛋白I(ANX-I)ELISA套組(目錄號MBS704042,批號N10140947,標準偵測範圍:0.312-20ng/mL;靈敏度:0.078ng/mL)係來自MyBioSource,Inc.,San Diego,CA。 Human Gas6 ELISA kit (Cat. No. SK00098-01, batch number 20111218) and human SP-D ELISA Kit (Cat. No. SK00457-01, batch number 20111135) were from Aviscora Bioscience, Santa Clara, CA. The standard detection range of the human Gas6 ELISA kit is 62.5-8000pg / mL, and the sensitivity is 31pg / ml. The standard detection range of the human SP-D ELISA kit is 78-5000pg / mL, and the sensitivity is 30pg / ml. Human MFG-E8 ELISA kit (Cat. No. DFGE80, Lot No. 307254, Standard detection range: 62.5-4000pg / mL; Sensitivity: 4.04pg / mL), Human TSP-1 ELISA kit (Cat. No. DTSP10, Lot No. 307182, Standard Detection range: 7.81-500ng / mL; sensitivity: 0.355ng / mL), human TSP-2 ELISA kit (catalogue number DTSP10, batch number 307266, standard detection range: 0.31-20ng / mL; sensitivity: 0.025ng / mL ) Is from R & D Systems, Inc., Minneapolis, MN. The apolipoprotein H human ELISA kit (catalog number ab108814, batch number GR126938, standard detection range: 0.625-40ng / mL, sensitivity: 0.6ng / mL) was from Abcam, Cambridge, MA. Human Annexin I (ANX-I) ELISA kit (catalog number MBS704042, batch number N10140947, standard detection range: 0.312-20ng / mL; sensitivity: 0.078ng / mL) is from MyBioSource, Inc., San Diego, CA .
hUTC、ARPE-19、及NHDF條件培養基之製備:在第1天,將hUTC、ARPE-19、及NHDF分別以10,000個存活細胞/cm2接種於T75細胞培養瓶中之15mL hUTC生長培養基(DMEM低葡萄糖+15% FBS+4mM L-麩醯胺酸)中。於37℃下、5% CO2培養器中培養24h。在第2天,吸出培養基且補充18mL DMEM/F12完 全培養基(DMEM:F12培養基+10% FBS+Pen(50U/ml)/Strep(50μg/ml))。再將細胞培養48h。亦將單獨對照培養基(DMEM:F12培養基+10% FBS+Pen(50U/ml)/Strep(50μg/ml))培養48h。在第4天,收集細胞培養物上清液及對照培養基,並將其在4℃下以250g離心5min,以0.5mL/管等分於凍存管中,且立即冷凍於-70℃冰箱。冷凍樣本係經解凍以供ELISA使用。 Preparation of conditioned medium for hUTC, ARPE-19 , and NHDF : On day 1, hUTC, ARPE-19, and NHDF were inoculated into 15mL hUTC growth medium (DMEM) in T75 cell culture flasks at 10,000 viable cells / cm 2 respectively Low glucose + 15% FBS + 4mM L-glutamic acid). Incubate at 37 ° C in a 5% CO 2 incubator for 24h. On the second day, the medium was aspirated and supplemented with 18 mL of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50 U / ml) / Strep (50 μg / ml)). The cells were cultured for 48h. A separate control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)) was also cultured for 48h. On Day 4, the cell culture supernatant and control medium were collected and centrifuged at 250g for 5 min at 4 ° C, aliquoted in 0.5mL / tube in cryopreservation tubes, and immediately frozen in a -70 ° C refrigerator. Frozen samples were thawed for ELISA.
每48h每百萬個hUTC細胞分泌77.2ng MFG-E8,相較於每48h每百萬個ARPE-19及NHDF細胞分別分泌7.6ng及17.5ng MFG-E8(圖12A)。hUTC條件培養基中MFG-E8之濃度為15.5ng/mL。對照培養基中MFG-E8之量不可偵測。(圖12E)。 77.2ng MFG-E8 is secreted per million hUTC cells every 48h, compared to 7.6ng and 17.5ng MFG-E8 secreted per million ARPE-19 and NHDF cells every 48h (Figure 12A). The concentration of MFG-E8 in hUTC conditioned medium is 15.5 ng / mL. The amount of MFG-E8 in the control medium is undetectable. (Figure 12E).
每48h每百萬個hUTC細胞分泌352.8pg Gas6,相較於ARPE-19分泌183.9pg及NHDF分泌1101pg(圖12B)。hUTC條件培養基中Gas6之濃度為70.6pg/mL。對照培養基中Gas6之量不可偵測。(圖11G)。 Every 48h, 352.8 pg of Gas6 was secreted per million hUTC cells, compared with 183.9 pg of ARPE-19 and 1101 pg of NHDF (Figure 12B). The concentration of Gas6 in hUTC conditioned medium is 70.6 pg / mL. The amount of Gas6 in the control medium is undetectable. (Figure 11G).
每48h每百萬個hUTC細胞分泌759.2ng TSP-1,相較於每48h每百萬個ARPE-19細胞分泌4744.68ng TSP-1及NHDF細胞分泌2487.55ng TSP-1(圖12C)。hUTC條件培養基中TSP-1之濃度為151.8ng/mL。對照培養基中偵測出4.0ng/mL TSP-1。(圖12E)。 Every 48h, 759.2ng TSP-1 was secreted per million hUTC cells, compared with 4748.68ng TSP-1 secreted per million ARPE-19 cells per 48h and 2487.55ng TSP-1 secreted by NHDF cells (Figure 12C). The concentration of TSP-1 in hUTC conditioned medium is 151.8ng / mL. 4.0ng / mL TSP-1 was detected in the control medium. (Figure 12E).
每48h每百萬個hUTC細胞分泌44.2ng TSP-2,相較於NHDF分泌30ng TSP-2。每48h每百萬個ARPE-19細胞釋放可忽略量0.08ng TSP-2(圖12D)。hUTC條件培養基中TSP-2之濃度為8.8ng/mL。對照培養基中偵測出痕量TSP-2(0.02ng/mL)。(圖12E)。 Every 48h, 44.2ng TSP-2 was secreted per million hUTC cells, compared to 30ng TSP-2 secreted by NHDF. Every 48h, a negligible amount of 0.08ng TSP-2 was released per million ARPE-19 cells (Figure 12D). The concentration of TSP-2 in hUTC conditioned medium is 8.8 ng / mL. Trace amounts of TSP-2 (0.02ng / mL) were detected in the control medium. (Figure 12E).
hUTC、ARPE-19及NHDF之條件培養基中載脂蛋白H之水準類似於對照培養基中之水準(6.8ng/mL)。hUTC、ARPE-19及NHDF條件培養基以及對照培養基中SP-D及膜聯蛋白I之水準低於ELISA之偵測界限(分別為<30pg/mL及<78pg/mL)。該些細胞不分泌這兩種蛋白質,或分泌水準低於偵測界限。 The level of apolipoprotein H in the conditioned medium of hUTC, ARPE-19 and NHDF is similar to the level in the control medium (6.8ng / mL). The levels of SP-D and Annexin I in conditioned medium of hUTC, ARPE-19 and NHDF and control medium were below the detection limit of ELISA (<30pg / mL and <78pg / mL, respectively). These cells do not secrete these two proteins, or the secretion level is below the detection limit.
在hUTC條件培養基中所檢測之橋分子及其濃度之概述列舉於表3-2中。 A summary of the bridge molecules and their concentrations detected in hUTC conditioned medium are listed in Table 3-2.
在所量測之橋分子中,MFG-E8、Gas6、TSP-1及TSP-2係涉及RCS RPE細胞中由hUTC所媒介之吞噬救援之候選橋分子。 Among the measured bridge molecules, MFG-E8, Gas6, TSP-1 and TSP-2 are candidate bridge molecules involved in hCSC-mediated phagocytosis rescue in RCS RPE cells.
經橋分子調理之ROS之結合可活化整合素及RTK傳訊路徑,其可補償Mertk傳訊之不存在,且引起吞噬之救援。 The combination of ROS regulated by the bridge molecule can activate the integrin and RTK communication path, which can compensate for the absence of Mertk communication and cause phagocytosis rescue.
hUTC條件培養基對吞噬ROS之直接作用係藉由對RCS RPE細胞饋以經hUTC條件培養基預培養之ROS來檢測。(US 2010/0272803)。失養性RPE細胞之吞噬係經完全救援。此處,研究存在於或經分泌於hUTC條件培養基中之橋分子之作用。目前,ROS上所識別之唯一「吃我」信號為磷脂醯絲胺酸(PS)(Finnemann等人,PNAS,2012;109(21):8145-8148)。 The direct effect of hUTC conditioned medium on phagocytosis of ROS was detected by feeding RCS RPE cells with ROS pre-cultured with hUTC conditioned medium. (US 2010/0272803). The phagocytosis of dystrophic RPE cells is completely rescued. Here, the role of bridge molecules present or secreted in hUTC conditioned medium is studied. Currently, the only "eat me" signal recognized on ROS is phospholipid amide serine (PS) (Finnemann et al., PNAS , 2012; 109 (21): 8145-8148).
用於RPE分離、RPE細胞之初代培養、RPE細胞之磺醯羅丹明染色、大鼠ROS之分離、ROS之FITC染色、吞噬檢定、成像和定量、檢定接受標準、及相對吞噬水準之程序係描述於實例1中。 The procedures for RPE isolation, primary culture of RPE cells, sulforadamine staining of RPE cells, isolation of rat ROS, FITC staining of ROS, phagocytosis assay, imaging and quantification, acceptance criteria for assay, and relative phagocytosis level are described In Example 1.
hUTC條件培養基(CM):hUTC CM3係用於本研究。在第1天,將hUTC以10,000個存活細胞/cm2接種於T75細胞培養瓶中之hUTC生長培養基(DMEM低葡萄糖+15% FBS+4mM L-麩醯胺酸)中。於37℃下、5% CO2培養器中培養24小時。在第2天,吸出培養基且補充21mL DMEM/F12完全培養基(DMEM:F12培養基+10% FBS+Pen(50U/ml)/Strep(50μg/ml))。再培養細胞48小時。亦將單獨對照培養基(DMEM:F12培養基+10% FBS+Pen(50 U/ml)/Strep(50μg/ml))培養48h。在第4天,收集細胞培養物上清液及對照培養基,並將其在RT下以250g離心5min,以3mL/管等分於凍存管中,且立即冷凍於-70℃冰箱。 hUTC conditioned medium ( CM ): hUTC CM3 is used in this study. On day 1, hUTC was seeded at 10,000 viable cells / cm 2 in hUTC growth medium (DMEM low glucose + 15% FBS + 4 mM L-glutamic acid) in T75 cell culture flasks. Incubate at 37 ° C in a 5% CO 2 incubator for 24 hours. On the second day, the medium was aspirated and supplemented with 21 mL of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50 U / ml) / Strep (50 μg / ml)). The cells were incubated for another 48 hours. A separate control medium (DMEM: F12 medium + 10% FBS + Pen (50 U / ml) / Strep (50 μg / ml)) was also cultured for 48 h. On the 4th day, the cell culture supernatant and control medium were collected and centrifuged at 250g for 5 min at RT, aliquoted in 3mL / tube into cryovials, and immediately frozen in a -70 ° C refrigerator.
重組人類橋分子:重組人類MFG-E8(目錄號2767-MF-050,批號MPP2012061)、重組人類Gas6(目錄號885-GS-050,批號GNT5013011)、重組人類TSP-1(目錄號3074-TH-050,批號MVF3613041)、重組人類TSP-2(目錄號1635-T2-050,批號HUZ1713021)全部皆獲自R&D Systems,Inc.,Minneapolis,MN。個別蛋白質儲備液之重構係根據供應商之資料表:重組人類MFG-E8、TSP-1及TSP-2分別以100μg/mL於無菌PBS中重構。重組人類Gas6以100μg/mL重構於無菌水中。經重構之儲備液經等分且冷凍於-70℃冰箱。 Recombinant human bridge molecules: recombinant human MFG-E8 (catalog number 2767-MF-050, batch number MPP2012061), recombinant human Gas6 (catalog number 885-GS-050, batch number GNT5013011), recombinant human TSP-1 (catalog number 3074-TH -050, batch number MVF3613041), recombinant human TSP-2 (catalogue number 1635-T2-050, batch number HUZ1713021) were all obtained from R & D Systems, Inc., Minneapolis, MN. The reconstitution of individual protein stock solutions was based on the supplier's data sheet: recombinant human MFG-E8, TSP-1 and TSP-2 were reconstituted in sterile PBS at 100 μg / mL, respectively. Recombinant human Gas6 was reconstituted in sterile water at 100 μg / mL. The reconstituted stock solution was aliquoted and frozen in the refrigerator at -70 ° C.
橋分子對RCS RPE細胞吞噬之作用:ROS係在37℃下以對照培養基(DMEM+10% FBS)或CM3於CO2細胞培養器中預培養24h。同時,ROS係於對照培養基中以各種濃度之人類重組MFG-E8、Gas6、TSP-1、或TSP-2在37℃下於CO2細胞培養器中預培養24h。在培養之後,將ROS離心沉降而不清洗,再懸浮於MEM5中,且在MEM5存在之情況下饋至失養性RPE細胞以進行吞噬檢定。至於對照組,將單獨正常RPE或單獨失養性RPE培養於MEM20中,隨後在未處理ROS(再懸浮於MEM20中且饋至RPE細胞)存在下更換成MEM5以進行吞噬檢定。 Effect of bridge molecule on phagocytosis of RCS RPE cells: ROS line was pre-cultured in CO 2 cell incubator with control medium (DMEM + 10% FBS) or CM3 at 37 ℃ for 24h. Meanwhile, ROS was pre-cultured in various concentrations of human recombinant MFG-E8, Gas6, TSP-1, or TSP-2 in a CO 2 cell culture incubator at 37 ° C for 24h. After culturing, ROS were centrifuged to settle without washing, resuspended in MEM5, and fed to the dystrophic RPE cells in the presence of MEM5 for phagocytosis assay. As for the control group, normal RPE alone or deprived RPE alone was cultured in MEM20, and then replaced with MEM5 in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) for phagocytosis assay.
RTK配體對RCS PRE細胞吞噬之作用:RCS RPE係與重組人類BDNF、HGF、及GDNF個別地培養24小時,且隨後添加OS以用於吞噬檢定。經hUTC CM培養之RCS RPE係用作陽性對照。 Effect of RTK ligand on phagocytosis of RCS PRE cells: RCS RPE was cultured with recombinant human BDNF, HGF, and GDNF individually for 24 hours, and then OS was added for phagocytosis assay. The RCS RPE cultured with hUTC CM was used as a positive control.
siRNA剔除:針對人類BDNF、HGF、GDNF、MFG-E8、Gas6、TSP-1、及TSP-2之On-TARGETplus人類siRNA-SMARTpool、以及ON-TARGETplus Non-targeting pool(淩亂siRNA池)係購自GE Dharmacon(Lafayette,CO)。使用DharmaFECT轉染試劑(GE Dharmacon),將25nM之各siRNA池分別併入hUTC。 siRNA Removal: On-TARGETplus human siRNA-SMARTpool and ON-TARGETplus Non-targeting pool for human BDNF, HGF, GDNF, MFG-E8, Gas6, TSP-1, and TSP-2 were purchased from GE Dharmacon (Lafayette, CO). Using DharmaFECT transfection reagent (GE Dharmacon), each siRNA pool of 25 nM was incorporated into hUTC.
免疫螢光染色之抗體:橋分子(人類MFG-E8、Gas6、TSP-1、及TSP-2)之未共軛單株抗體、以及小鼠IgG2A與IgG2B同型對照抗體係獲自R&D Systems,Inc.,Minneapolis,MN。此等抗體由Life Technologies(Eugene,OR)來與Alexa Fluor 488螢光團共軛。未共軛之單株抗視紫質抗體(EMD Millipore Corp.,Temecula CA)由Life Technologies(Eugene,OR)來與Alexa Fluor 568共軛。未共軛之小鼠IgG2b,x同型對照抗體係獲自Biolegend公司(San Diego,CA)且由Life Technologies(Eugene,OR)來與Alexa Fluor 488螢光團共軛。其係作為Alexa Fluor 568共軛抗視紫質抗體之同型對照抗體。Alexa Fluor 488共軛小鼠IgG2A係作為Alexa Fluor 488共軛抗人類MFG-E8、Gas6、或TSP-2抗體之同型對照抗體。Alexa Fluor 488共軛小鼠IgG2B係作為Alexa Fluor 488共軛人類TSP-1抗體之同型對照抗體。 Immunofluorescent stained antibodies: unconjugated monoclonal antibodies of bridge molecules (human MFG-E8, Gas6, TSP-1, and TSP-2), and mouse IgG2A and IgG2B isotype control antibody systems were obtained from R & D Systems, Inc ., Minneapolis, MN. These antibodies were conjugated to Alexa Fluor 488 fluorophore by Life Technologies (Eugene, OR). The unconjugated single anti-rhodopsin antibody (EMD Millipore Corp., Temecula CA) was conjugated to Alexa Fluor 568 by Life Technologies (Eugene, OR). The unconjugated mouse IgG2b, x isotype control antibody system was obtained from Biolegend Corporation (San Diego, CA) and conjugated with Alexa Fluor 488 fluorophore by Life Technologies (Eugene, OR). It is used as an isotype control antibody for Alexa Fluor 568 conjugated anti-rhodopsin antibody. Alexa Fluor 488 conjugated mouse IgG2A line is used as an isotype control antibody for Alexa Fluor 488 conjugated anti-human MFG-E8, Gas6, or TSP-2 antibody. Alexa Fluor 488 conjugated mouse IgG2B line is used as isotype control antibody of Alexa Fluor 488 conjugated human TSP-1 antibody.
免疫螢光:將10×106個OS細胞在37℃下於1mL hUTC CM、1mL對照培養基、或1mL含有124ng/mL MFG-E8、8.75ng/mL Gas6、1.2μg/mL TSP-1、或238ng/mL TSP-2之對照培養基中培養24小時。使OS成團塊、清洗且包埋於Tissue-Tek O.C.T化合物(Sakura Finetek USA,Inc.,Torrance,CA)中。使用低溫恆溫器(Leica CM1950,Leica Microsystems,Inc.,Buffalo Grove,Illinois)以獲得10 iAm系列切片。將該些切片轉移至玻片上用於免疫螢光染色。將OS片之圈起位置在室溫下用阻斷緩衝液(blocking buffer)(10%(v/v)山羊血清、1%(v/v)BSA、及0.1%(v/v)Triton×100於PBS中)處理1小時,且隨後在4℃下用Alexa Fluor 568共軛抗視紫質抗體及Alexa Fluor 488共軛抗MFG-E8、抗Gas6、抗TSP-1、抗TSP-2、或小鼠IgG2A或IgG2B同型對照抗體雙重染色2小時。用PBS清洗三次之後,將切片固定於Vectashield封片劑(Vector Laboratories,Inc.,Burlingame,CA)中,並以裝備有落射螢光的Zeiss Photomicroscope III(Carl Zeiss,Oberkochen,Germany)評估。使用Kodak 290數位照相機擷取影像且使用Kodak Microscopy Documentation System 290 Photoshop影像分析軟體(Eastman Kodak,Rochester,NY)分析。以250x放大率用適當的過濾器獲得影像。 Immunofluorescence: 10 × 10 6 OS cells at 37 ° C in 1 mL hUTC CM, 1 mL control medium, or 1 mL containing 124 ng / mL MFG-E8, 8.75 ng / mL Gas6, 1.2 μg / mL TSP-1, or Incubate for 24 hours in 238ng / mL TSP-2 control medium. The OS was agglomerated, washed and embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Inc., Torrance, CA). A cryostat (Leica CM1950, Leica Microsystems, Inc., Buffalo Grove, Illinois) was used to obtain 10 iAm series of slices. These slices were transferred to slides for immunofluorescence staining. Use the blocking buffer (10% (v / v) goat serum, 1% (v / v) BSA, and 0.1% (v / v) Triton × 100 in PBS) for 1 hour, and then at 4 ° C with Alexa Fluor 568 conjugated anti-rhodopsin antibody and Alexa Fluor 488 conjugated anti-MFG-E8, anti-Gas6, anti-TSP-1, anti-TSP-2, Or mouse IgG 2 A or IgG2 B isotype control antibody double stained for 2 hours. After washing three times with PBS, the sections were fixed in Vectashield mounted tablets (Vector Laboratories, Inc., Burlingame, CA) and evaluated with Zeiss Photomicroscope III (Carl Zeiss, Oberkochen, Germany) equipped with epi-fluorescence. Images were captured using a Kodak 290 digital camera and analyzed using Kodak Microscopy Documentation System 290 Photoshop image analysis software (Eastman Kodak, Rochester, NY). The image is obtained with an appropriate filter at 250x magnification.
如圖13A與圖13B(實驗1)及圖13C與13D(實驗2)中所示,未處理失養性RPE細胞相較於正常RPE細胞減少吞噬。 在檢定期間不存在hUTC條件培養基之情況下,用hUTC條件培養基預培養失養性RPE細胞完全救援吞噬。當hUTC條件培養基存在於吞噬檢定期間時,不管失養性RPE細胞是否經hUTC條件培養基預處理,皆觀察到更為穩固增強之吞噬。饋以經hUTC條件培養基預處理之ROS的失養性RPE細胞在吞噬檢定期間不存在hUTC條件培養基下顯示吞噬之恢復。 As shown in FIGS. 13A and 13B (Experiment 1) and FIGS. 13C and 13D (Experiment 2), untreated defermented RPE cells reduced phagocytosis compared to normal RPE cells. In the absence of hUTC conditioned medium during the test period, precultivation of dystrophic RPE cells with hUTC conditioned medium completely rescues phagocytosis. When hUTC conditioned medium is present during the phagocytosis assay, whether or not the defermented RPE cells are pretreated with hUTC conditioned medium, a more stable and enhanced phagocytosis is observed. The dystrophic RPE cells fed with ROS pretreated with hUTC conditioned medium showed recovery of phagocytosis in the absence of hUTC conditioned medium during the phagocytosis assay.
將失養性RPE細胞饋以用各種濃度之MFG-E8(15.5ng/mL;31ng/mL;62ng/mL;124ng/mL)、Gas6(70pg/mL;350pg/mL;1750pg/mL;8750pg/mL)、TSP-1(152ng/mL;304ng/mL;608ng/mL;1216ng/mL)、或TSP-2(8.8ng/mL;26.4ng/mL;79.2ng/mL;237.6ng/mL)預培養之ROS,並檢定其吞噬(圖14A至圖14D)。每個樣本觀察十次。吞噬ROS藉由對RCS RPE細胞饋以經MFG-E8、Gas6、TSP-1或TSP-2預培養之ROS而救援。 The dystrophic RPE cells were fed with various concentrations of MFG-E8 (15.5ng / mL; 31ng / mL; 62ng / mL; 124ng / mL), Gas6 (70pg / mL; 350pg / mL; 1750pg / mL; 8750pg / mL ), TSP-1 (152ng / mL; 304ng / mL; 608ng / mL; 1216ng / mL), or TSP-2 (8.8ng / mL; 26.4ng / mL; 79.2ng / mL; 237.6ng / mL) pre-culture ROS, and tested for phagocytosis (Figure 14A to Figure 14D). Each sample was observed ten times. Phagocytosis of ROS is rescued by feeding RCS RPE cells with ROS pre-cultured with MFG-E8, Gas6, TSP-1 or TSP-2.
MFG-E8、Gas6、TSP-1、及TSP-2中之各者劑量依賴性增加RCS RPE細胞中之吞噬水準(圖14E至圖14H)。類似地,BDNF、HGF及GDNF劑量依賴性增加RCS RPE細胞中之吞噬失準,其中HGF即使在最低劑量下之作用仍最強。當以較高濃度施加時,BDNF、HGF及GDNF能夠救援RCS RPE中之吞噬(圖14I至J)。此等結果顯示,重組RTK配體及橋分子蛋白可模擬hUTC CM之作用且恢復RCS RPE吞噬,且涉及RCS RPE中hUTC媒介之吞噬救援。 Each of MFG-E8, Gas6, TSP-1, and TSP-2 dose-dependently increased the level of phagocytosis in RCS RPE cells (Figures 14E to 14H). Similarly, BDNF, HGF and GDNF dose-dependently increased phagocytic misalignment in RCS RPE cells, with HGF still having the strongest effect even at the lowest dose. When applied at higher concentrations, BDNF, HGF and GDNF were able to rescue phagocytosis in RCS RPE (Figures 14I to J). These results show that the recombinant RTK ligand and bridge protein can mimic the role of hUTC CM and restore RCS RPE phagocytosis, and involve hUTC-mediated phagocytosis rescue in RCS RPE.
BDNF、HGF、GDNF、MFG-E8、Gas6、TSP-1及TSP-2係藉由siRNA媒介基因靜默於hUTC中剔除。不靶向任何基因之淩亂siRNA池係用來作為基因剔除對照。各因子之剔除效率藉由量測細胞培養物上清液中各因子之水準來檢測,該些細胞培養物上清液係收集自經siRNA轉染之hUTC(圖15A)。模擬或淩亂siRNA轉染對此等因子的hUTC分泌無作用。靶向MFG-E8、TSP-1、TSP-2、及HGF之siRNA產生幾乎100%剔除效率;BDNF及GDNF分別觀察到80%及65%剔除(圖15A)。hUTC中靶向Gas6之siRNA不會作用(數據未顯示)。CM係自siRNA轉染之hUTC生產且施加至RCS RPE以識別RTK配體與橋分子剔除之作用。將RCS RPE用生產自經靶向BDNF、HGF或GDNF之siRNA轉染的hUTC之CM培養(圖15B),或饋以生產自經靶向MFG-E8、TSP-1或TSP-2之siRNA轉染的hUTC之CM預處理的OS(圖15C)。製備自未轉染及淩亂siRNA轉染之hUTC之CM係用來作為剔除對照CM。RTK配體各者之個別剔除相較於剔除對照CM廢除hUTC CM對吞噬救援之作用(圖15B)。剔除橋分子中之各者降低RCS RPE對OS之吞噬(圖15C)。此等RTK配體與橋分子為RCS RPE中hUTC媒介之吞噬救援所需。 BDNF, HGF, GDNF, MFG-E8, Gas6, TSP-1 and TSP-2 were eliminated in hUTC by siRNA-mediated gene silencing. A messy siRNA pool that does not target any genes is used as a gene knockout control. The removal efficiency of each factor was detected by measuring the level of each factor in the cell culture supernatant, which was collected from hUTC transfected with siRNA (Figure 15A). Simulated or messy siRNA transfection has no effect on the secretion of hUTC from these factors. SiRNAs targeting MFG-E8, TSP-1, TSP-2, and HGF produced almost 100% rejection efficiency; BDNF and GDNF were observed to be 80% and 65% removed, respectively (Figure 15A). siRNAs targeting Gas6 in hUTC will not work (data not shown). CM is produced from hUTC transfected with siRNA and applied to RCS RPE to recognize the role of RTK ligand and bridge molecule knockout. Incubate RCS RPE with CM produced from hUTC transfected with siRNA targeting BDNF, HGF or GDNF (Figure 15B), or feed with siRNA produced from MFG-E8, TSP-1 or TSP-2 CM pretreated OS of stained hUTC (Figure 15C). The CM prepared from untransfected and messy siRNA transfected hUTC was used as a control CM. The individual knockout of each RTK ligand abolished the effect of hUTC CM on phagocytosis rescue compared to the knockout control CM (Figure 15B). Removal of each of the bridge molecules reduces the phagocytosis of OS by RCS RPE (Figure 15C). These RTK ligands and bridge molecules are required for hUTC-mediated phagocytosis rescue in RCS RPE.
每一個別橋分子與視紫質之雙染色確保OS受到評估。視紫質係位於光受體OS中之視色素且為OS染色之標誌(Szabo K等人,Cell Tissue Res.2014;356(1):49-63)。經個別重組人類橋分子預培養之視紫質染色(Alexa Fluor 568共軛,紅色)粒子經四種 橋分子抗體(Alexa Fluor 488共軛,綠色)之各者染色呈陽性,但Alexa Fluor 488共軛小鼠IgG2A或IgG2B同型對照抗體染色則不呈陽性(圖16A)。經hUTC CM培養之OS獲得類似結果(圖16B),然而經對照培養基培養之OS則未觀察到任何橋分子染色(圖16C)。抗視紫質抗體之特異性藉由以Alexa Fluor 568共軛抗視紫質抗體及Alexa Fluor 488共軛小鼠IgG2b,x同型對照抗體雙重染色OS粒子來確認。僅抗視紫質抗體染色之OS呈陽性(圖16D)。hUTC CM中之橋分子MFG-E8、Gas6、TSP-1及TSP-2與視紫質共定位於OS上證明橋分子結合至OS。 Double staining of each individual bridge molecule and rhodopsin ensures that OS is evaluated. Rhodopsin is a visual pigment located in the photoreceptor OS and is a sign of OS staining (Szabo K et al., Cell Tissue Res. 2014; 356 (1): 49-63). The rhodopsin staining (Alexa Fluor 568 conjugated, red) particles pre-cultured with individual recombinant human bridge molecules were stained positive by each of the four bridge molecule antibodies (Alexa Fluor 488 conjugated, green), but Alexa Fluor 488 Conjugated mouse IgG2A or IgG2B isotype control antibody staining was not positive (Figure 16A). Similar results were obtained for OS cultured with hUTC CM (Figure 16B), however, OS bridge cultured with the control medium did not observe any bridge molecule staining (Figure 16C). The specificity of anti-rhodopsin antibodies was confirmed by double staining of OS particles with Alexa Fluor 568 conjugated anti-rhodopsin antibody and Alexa Fluor 488 conjugated mouse IgG2b, x isotype control antibody. Only OS stained with anti-rhodopsin antibody was positive (Figure 16D). The bridge molecules MFG-E8, Gas6, TSP-1 and TSP-2 in hUTC CM co-localize with rhodopsin on the OS, proving that the bridge molecules bind to the OS.
氧化應力可影響視網膜色素上皮之健康。研究hUTC及hUTC條件培養基對改良暴露於氧化損傷之RPE細胞之健康的作用。 Oxidative stress can affect the health of retinal pigment epithelium. To study the effect of hUTC and hUTC conditioned medium on improving the health of RPE cells exposed to oxidative damage.
過氧化氫(H2O2)、結晶紫及噻唑基藍四唑鎓溴化物(MTT)獲自Sigma-Aldrich(St Louis,MO)。 Hydrogen peroxide (H 2 O 2 ), crystal violet and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St Louis, MO).
哈姆F10培養基、青黴素-鏈黴素溶液(5000個單位/mL青黴素/5000μg/mL鏈黴素)、胰蛋白酶-EDTA溶液(0.05%)、低葡萄糖DMEM、L-麩醯胺酸200mM獲自Life Technologies。 Ham F10 medium, penicillin-streptomycin solution (5000 units / mL penicillin / 5000 μg / mL streptomycin), trypsin-EDTA solution (0.05%), low glucose DMEM, L-glutamic acid 200 mM were obtained from Life Technologies.
Hyclone FBS及甲醛購自Thermo Scientific。異丙醇、冰乙酸及鹽酸購自Fisher Scientific(Pittsburgh,PA)。乙醇獲自Decon Labs Inc.(King of Prussia,PA)。PBS獲自Lonza(South Plainfield,NJ)。 Hyclone FBS and formaldehyde were purchased from Thermo Scientific. Isopropyl alcohol, glacial acetic acid and hydrochloric acid were purchased from Fisher Scientific (Pittsburgh, PA). Ethanol was obtained from Decon Labs Inc. (King of Prussia, PA). PBS was obtained from Lonza (South Plainfield, NJ).
ARPE生長培養基:DMEM(具有4.5g/L葡萄糖及丙酮酸鈉而無L-麩醯胺酸及酚紅)(Mediatech,Inc.A Corning Subsidiary,Manassas,VA)補充有5%或10%熱失活胎牛血清(FBS,Life Technologies,Grand Island,NY)、1X最低必需培養基-非必需胺基酸(MEM-NEAA,Life Technologies)、及0.01mg/mL建它黴素試劑溶液(Life Technologies)。 ARPE growth medium: DMEM (with 4.5g / L glucose and sodium pyruvate without L-glutamic acid and phenol red) (Mediatech, Inc. A Corning Subsidiary, Manassas, VA) supplemented with 5% or 10% heat loss Live fetal bovine serum (FBS, Life Technologies, Grand Island, NY), 1X minimum essential medium-non-essential amino acids (MEM-NEAA, Life Technologies), and 0.01 mg / mL itamycin reagent solution (Life Technologies) .
含有10μM A2E之5% ARPE生長培養基:DMEM(Mediatech,Inc.A Corning Subsidiary)補充有5%熱失活FBS(Life Technologies)、1X MEM-NEAA(Life Technologies)、0.01mg/mL建它黴素試劑溶液(Life Technologies)、及10μM A2E(由Janet Sparrow博士之實驗室製備)。 5% ARPE growth medium containing 10 μM A2E : DMEM (Mediatech, Inc. A Corning Subsidiary) supplemented with 5% heat-inactivated FBS (Life Technologies), 1X MEM-NEAA (Life Technologies), 0.01 mg / mL citracin Reagent solution (Life Technologies), and 10 μM A2E (prepared by Dr. Janet Sparrow's laboratory).
hUTC完全培養基:DMEM低葡萄糖(Life Technologies)補充有15% Hyclone® FBS(Thermo Scientific,Logan,Utah)及4mML-麩醯胺酸(Life Technologies)。 hUTC complete medium: DMEM low glucose (Life Technologies) supplemented with 15% Hyclone® FBS (Thermo Scientific, Logan, Utah) and 4mML-glutamic acid (Life Technologies).
hUTC FBS培養基:DMEM(Mediatech,Inc.)補充有5%或10%熱失活FBS(Life Technologies)、1X MEM-NEAA(Life Technologies)、0.01mg/mL建它黴素試劑溶液(Life Technologies)、及4mM L-麩醯胺酸(Mediatech,Inc.)。 hUTC FBS medium: DMEM (Mediatech, Inc.) supplemented with 5% or 10% heat-inactivated FBS (Life Technologies), 1X MEM-NEAA (Life Technologies), 0.01 mg / mL itanomycin reagent solution (Life Technologies) , And 4mM L-glutamic acid (Mediatech, Inc.).
hUTC條件培養基:將hUTC(研究庫NB12898P6,PDL20)在37℃、5% CO2下以5000個細胞/cm2接種於2個T75培養瓶中之hUTC完全培養基(15mL)中。接種後24小時,將培養基自各培養瓶移除,且將細胞以15mL 1X達爾伯克磷酸鹽緩衝液(DPBS)清洗3次。在第三次清洗之後,將15mL 5%或10% FBS hUTC培養基添加至各瓶。亦將十五mL 5%或10% FBS hUTC培養基添加至2個空T75瓶中並充當對照組。將所有瓶放回37℃、5% CO2 48小時。48小時之後,將培養基自各培養瓶移除,且在4℃下以250×g離心5分鐘。將培養基放置於冰上且隨後等分且儲存在-80℃下。 hUTC conditioned medium: hUTC (Research Library NB12898P6, PDL20) was inoculated into hUTC complete medium (15 mL) in 2 T75 culture flasks at 37 ° C and 5% CO 2 at 5000 cells / cm 2 . Twenty-four hours after seeding, the medium was removed from each flask, and the cells were washed 3 times with 15 mL of 1X Dulbecco's phosphate buffered saline (DPBS). After the third wash, 15 mL of 5% or 10% FBS hUTC medium was added to each bottle. Fifteen mL of 5% or 10% FBS hUTC medium was also added to 2 empty T75 bottles and served as a control group. Return all bottles to 37 ° C, 5% CO 2 for 48 hours. After 48 hours, the medium was removed from each flask and centrifuged at 250 × g for 5 minutes at 4 ° C. The medium was placed on ice and then aliquoted and stored at -80 ° C.
用於A2E研究之ARPE-19細胞培養:在第1天,將ARPE-19細胞以40,000個細胞/孔之密度接種於8孔NuncTM Lab-TekTM II腔室玻片(Nalge Nunc International Corporation,Rochester,NY)中最終體積300μL 10% ARPE生長培養基中。接種後24小時(第2天),將細胞上之培養基移除且以300μL 5% ARPE生長培養基替換。一週之後(第9天),再次將培養基移除且以新鮮5% ARPE生長培養基替換。在第14天,將培養基移除且以含有10μM A2E之新鮮5% ARPE生長培養基替換。在第17及21天,再次將培養基移除且以新鮮含A2E之培養基替換。在第24天,將含A2E之培養基移除且以新鮮5% ARPE生長培養基替換,且使細胞靜止五天。 ARPE-19 cell culture for A2E research: On day 1, ARPE-19 cells were seeded at a density of 40,000 cells / well on 8-well Nunc ™ Lab-Tek ™ II chamber slides (Nalge Nunc International Corporation, Rochester, NY) in a final volume of 300 μL of 10% ARPE growth medium. 24 hours after seeding (day 2), the medium on the cells was removed and replaced with 300 μL of 5% ARPE growth medium. One week later (Day 9), the medium was removed again and replaced with fresh 5% ARPE growth medium. On day 14, the medium was removed and replaced with fresh 5% ARPE growth medium containing 10 μM A2E. On days 17 and 21, the medium was removed again and replaced with fresh A2E-containing medium. On day 24, the A2E-containing medium was removed and replaced with fresh 5% ARPE growth medium, and the cells were quiescent for five days.
在第29天,將培養基自各孔移除且以250μL 5或10% hUTC條件培養基或對照培養基(未暴露於細胞之5%及10% FBS hUTC培養基)替換。在第32及35天,將條件培養基及對照培養基自細胞移除且以新鮮條件培養基或對照培養基替換。 On day 29, the medium was removed from each well and replaced with 250 μL of 5 or 10% hUTC conditioned medium or control medium (5% and 10% FBS hUTC medium not exposed to cells). On days 32 and 35, the conditioned medium and control medium were removed from the cells and replaced with fresh conditioned medium or control medium.
在第36天,將所有培養基自各孔移除且將細胞用1X DPBS清洗一次。稍後將200μL新鮮DPBS添加至各孔且將細胞暴露於由鎢絲鹵素來源遞送之430nm光20分鐘。在光暴露之後,將DPBS移除且進行細胞存活性檢定。 On day 36, all media was removed from each well and the cells were washed once with IX DPBS. Later, 200 μL of fresh DPBS was added to each well and the cells were exposed to 430 nm light delivered by a tungsten halogen source for 20 minutes. After light exposure, DPBS was removed and a cell viability test was performed.
用於A2E研究之MTT檢定:藉由代謝(MTT,3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓溴化物)比色微滴定檢定(Roche Diagnostics Corporation,Indianapolis,IN)量測細胞毒性。為進行MTT檢定,將20μL MTT標記試劑(Roche Diagnostics Corporation)添加至各孔中之0.2mL 5%培養基中。在培養4小時之後,將另一200μL增溶溶液添加至各孔用於隔夜培養。在以13,000rpm離心2分鐘之後,在570nm下以分光光度法量測上清液(SpectaMax MJ,Molecular Devices,Sunnyvale,CA)。還原MTT在570nm下之吸光度降低指出細胞存活性減弱。以Prism軟體分析數據。 MTT assay for A2E research: by metabolic (MTT, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) colorimetric microtitration assay ( Roche Diagnostics Corporation, Indianapolis, IN) measures cytotoxicity. For MTT assay, 20 μL of MTT labeling reagent (Roche Diagnostics Corporation) was added to 0.2 mL of 5% medium in each well. After culturing for 4 hours, another 200 μL of solubilizing solution was added to each well for overnight culture. After centrifugation at 13,000 rpm for 2 minutes, the supernatant was measured spectrophotometrically at 570 nm (SpectaMax MJ, Molecular Devices, Sunnyvale, CA). The reduced absorbance of reduced MTT at 570 nm indicates a decrease in cell viability. Analyze the data with Prism software.
用於A2E研究之Dead Red檢定:在藉由允許標記細胞凋亡細胞核之螢光排除檢定(fluorescence exclusion assay)標記之後,定量非存活細胞,允許標記細胞凋亡細胞核係由於在細胞死亡之後期階段細胞膜完整性喪失所致。在光暴露之後,將細胞放回至5% ARPE生長培養基中。在藍光暴露後八小時,將死亡細胞之細胞核用膜不透性染料Dead Red染色(Life Technologies;1/500稀釋,15min培養),並將所有細胞核用4’,6’-二胺基-2-苯基吲哚(DAPI) (Life Technologies)染色。簡言之,將細胞用預熱(37℃)的漢克斯氏平衡鹽溶液(1X)HBSS(Life Technologies)清洗兩次。將250μL Dead Red工作溶液(8μL Dead Red儲備液+4mL HBSS)在室溫下添加至各孔中15分鐘。在15分鐘之後,將細胞用HBSS清洗兩次。在室溫下將300μL 4%甲醛製備之1X DPBS添加至各孔中30分鐘。將細胞用1X DPBS清洗3次且在室溫下用DAPI(以1:300製備於1X DPBS)工作溶液培養5分鐘。將細胞用1X DPBS清洗3次。固定玻片且加蓋載玻片,且藉由計數各孔中照明區域內至少5個顯微鏡視野中DAPI染色及Dead-Red染色之細胞核來檢定重複物。值係表示為Dead-Red染色細胞核/DAPI染色細胞核×100。 Dead Red test for A2E research: After labeling by fluorescence exclusion assay allowing labeling of apoptotic nuclei, quantify non-surviving cells, allowing labeling of apoptotic nuclei due to the late stage of cell death Caused by loss of cell membrane integrity. After light exposure, the cells were put back into 5% ARPE growth medium. Eight hours after blue light exposure, the nuclei of dead cells were stained with membrane-impermeable dye Dead Red (Life Technologies; 1/500 dilution, 15 min incubation), and all nuclei were stained with 4 ', 6'-diamine-2 -Phenylindole (DAPI) (Life Technologies) staining. Briefly, cells were washed twice with preheated (37 ° C) Hanks' balanced salt solution (1X) HBSS (Life Technologies). 250 μL Dead Red working solution (8 μL Dead Red stock solution + 4 mL HBSS) was added to each well at room temperature for 15 minutes. After 15 minutes, the cells were washed twice with HBSS. 300 μL of 4% formaldehyde-made 1X DPBS was added to each well at room temperature for 30 minutes. The cells were washed 3 times with 1X DPBS and incubated with a working solution of DAPI (prepared in 1X DPBS at 1: 300) at room temperature for 5 minutes. The cells were washed 3 times with 1X DPBS. Slides were fixed and covered with slides, and duplicates were verified by counting DAPI stained and Dead-Red stained nuclei in at least 5 microscope fields in the illuminated area in each well. Values are expressed as Dead-Red stained nuclei / DAPI stained nuclei × 100.
用於H 2 O 2 研究之細胞培養:使ARPE-19細胞(美國菌種保存中心;Manassas,VA)在37℃、5% CO2下以單層形式生長於在T75瓶中之含有10% FBS及50個單位/mL青黴素/50μg/mL鏈黴素的哈姆F10培養基(Ham's F10 Medium)中。在與hUTC共培養之實驗中,ARPE19細胞係於T-75瓶中生長至80-90%長滿且稍後接種於24孔細胞培養板中。使ARPE-19細胞生長於10% FBS生長培養基中直至接種之後第3天,此時將培養基改變成基礎培養基(哈姆F10培養基補充有2% FBS及50個單位/mL青黴素/50μg/mL鏈黴素)。 Cell culture for H 2 O 2 research: ARPE-19 cells (American Bacteria Conservation Center; Manassas, VA) were grown in a single layer at 37 ° C and 5% CO 2 in a T75 flask containing 10% FBS and 50 units / mL penicillin / 50 μg / mL streptomycin in Ham's F10 Medium. In experiments co-cultivated with hUTC, ARPE19 cells were grown in T-75 flasks to 80-90% overgrown and later seeded in 24-well cell culture plates. ARPE-19 cells were grown in 10% FBS growth medium until day 3 after inoculation, at which time the medium was changed to basal medium (Hamm F10 medium supplemented with 2% FBS and 50 units / mL penicillin / 50 μg / mL chain Amycin).
將hUTC以5000個細胞/cm2在hUTC完全生長(低葡萄糖DMEM補充有15% FBS及4mM L-麩醯胺酸)中接種至細胞培養插入物(孔大小1μm)上24小時。將插入物轉移至生長於細 胞培養板72小時之ARPE-19細胞且於hUTC完全培養基中生長。將插入物移除且將製備於無血清哈姆F10培養基中之ARPE-19細胞以H2O2(0至1500μM)處理9小時。 HUTC was seeded onto cell culture inserts (well size 1 μm) in hUTC complete growth (low glucose DMEM supplemented with 15% FBS and 4 mM L-glutamic acid) at 5000 cells / cm 2 for 24 hours. The insert was transferred to ARPE-19 cells grown on cell culture plates for 72 hours and grown in hUTC complete medium. The insert was removed and ARPE-19 cells prepared in serum-free Ham F10 medium were treated with H 2 O 2 (0 to 1500 μM) for 9 hours.
用於H 2 O 2 研究之結晶紫細胞存活性檢定:藉由結晶紫攝取測定相對細胞存活性。在處理之後,將細胞固定於4%於PBS中之多聚甲醛中且於0.1%結晶紫、10%乙醇之溶液中染色。在用水清洗之後,將剩餘染色劑溶解於10%乙酸中且用微板讀數儀量測在550nm下之吸光度。 Crystal Violet Cell Viability Assay for H 2 O 2 research: Relative cell viability was determined by crystal violet uptake. After the treatment, the cells were fixed in 4% paraformaldehyde in PBS and stained in a solution of 0.1% crystal violet and 10% ethanol. After washing with water, the remaining stain is dissolved in 10% acetic acid and the absorbance at 550 nm is measured with a microplate reader.
用於H 2 O 2 研究之MTT檢定:將細胞在37℃下與0.25mg/mL於無血清培養基中之MTT培養3小時。隨後將培養基移除且添加酸性異丙醇(每1mL異丙醇1μL濃HCl)以增溶所產生之藍色甲(formazan)(MTT代謝產物)。藍色甲之密度係使用微板讀數儀在550nm下量測,背景波長為630nm。 MTT assay for H 2 O 2 research: Cells were incubated with MTT at 0.25 mg / mL in serum-free medium at 37 ° C for 3 hours. The medium was then removed and acidic isopropanol (1 μL concentrated HCl per 1 mL isopropanol) was added to solubilize the blue (formazan) (MTT metabolite). Blue armor The density is measured using a microplate reader at 550nm, and the background wavelength is 630nm.
hUTC條件培養基保護富含A2E之RPE細胞免於藍光誘導之損傷。ARPE-19存活性係於照射後藉由用膜不透性染料Dead Red標記細胞且用DAPI染色標記所有細胞核來評估。於數位影像中計數之細胞核提供存活與非存活細胞之百分比(圖17A至圖17B)。在430nm照明不存在下,10μM A2E對ARPE-19存活性無作用。用對照培養基培養且經歷430nm照明之細胞顯示高水準非存 活細胞(約50%)。相反地,用hUTC條件培養基處理導致非存活細胞之數目減少(約20%)。 The hUTC conditioned medium protects A2E-rich RPE cells from blue light-induced damage. ARPE-19 viability was evaluated after irradiation by labeling cells with the membrane-impermeable dye Dead Red and labeling all nuclei with DAPI staining. The nuclei counted in the digital image provide the percentage of viable and non-viable cells (Figure 17A-17B). In the absence of 430 nm illumination, 10 μM A2E had no effect on ARPE-19 viability. Cells cultured with control medium and subjected to 430 nm illumination showed high levels of non-viable cells (about 50%). Conversely, treatment with hUTC conditioned medium resulted in a reduction in the number of non-viable cells (about 20%).
亦藉由MTT檢定量測細胞存活性(圖17C至圖17D),其基於健康細胞將黃色四唑鎓鹽MTT切斷成紫色甲晶體之能力。甲之生產與培養物中存活細胞之數目成比例。用條件培養基處理且暴露於光之ARPE-19細胞顯示相較於添加10μM A2E且未暴露於光之ARPE-19細胞的存活性減少。用5或10% hUTC條件培養基處理之細胞顯示存活性高於暴露於對照培養基之細胞。 The cell viability was also quantified by MTT assay (Figures 17C to 17D), which cut off the yellow tetrazolium salt MTT into purple formazan based on healthy cells The power of crystals. A The production is proportional to the number of surviving cells in the culture. ARPE-19 cells treated with conditioned medium and exposed to light showed reduced viability compared to ARPE-19 cells added with 10 μM A2E and not exposed to light. Cells treated with 5 or 10% hUTC conditioned medium showed higher viability than cells exposed to control medium.
在總H2O2處理之後,藉由結晶紫及MTT檢定測定ARPE19細胞存活性(圖17E至圖17F)。與hUTC共培養之ARPE-19細胞相較於未處理之對照細胞顯示在用1500μM H2O2處理之後存活性得以改善。 After total H 2 O 2 treatment, ARPE19 cell viability was determined by crystal violet and MTT assay (Figure 17E to Figure 17F). ARPE-19 cells co-cultured with hUTC showed improved viability after treatment with 1500 μM H 2 O 2 compared to untreated control cells.
將在濕室中獲得的人類眼球於具有4% Pen-Strep(Life Technologies,青黴素-鏈黴素,10,000U/mL,目錄號15140122)之PBS中潤洗,並且在角膜緣處移除前節(若尚未移除)以將其打開。進行剖檢及照相之後,將視網膜移除,並將後極切成小片(約1cm)且在37C下培養於4ml 2%分散酶溶液(w/v,於DMEM中,25mM HEPES,200U/mL Pen-Strep)(Roche Diagnostic Corp,目錄號04942078001)中5分鐘。添加至少雙倍體積之DMEM(Life Technologies,目錄號12430054)、25mM HEPES(Sigma-Aldrich Corp.,目錄號H4034-100G)、200U/mL Pen-Strep,以停止培養。將組織放入100mm培養皿中,該培養皿具有足量新鮮DMEM、25mM HEPES、2% Pen-Strep,並且允許其在37℃下靜置6至12小時。將組織轉移至於培養皿中之RPMI培養基(Life Technologies,目錄號22400089),並藉由輕輕刮削或吸管尖的噴液,將RPE細胞自脈絡膜解開(teased off)。將細胞用HBSS(GIBCO,目錄號310-4170)潤洗2次,再懸浮於0.5ml 0.1%胰蛋白酶(pH 8.0,Life Technologies,目錄號25300054)中,並在37C下培養2分鐘。將細胞混合物用巴斯德吸量管(Pasteur pipette)吹打(triturated),直到RPE細胞分散為止,並添加5x過量之MEM/20% FBS(最低必需培養基,Lift Technologies,目錄號1095098;胎牛血清,Life Technologies,目錄號16000036)以停止酶處理。將細胞以1000 xg離心,再懸浮於適當體積的MEM/20(含有20% FBS之MEM)中,計數,且培養於適當容器(24孔盤,Thomas Scientific,目錄號6901A11;25ml瓶,VWR,目錄號29185-298)中。大致上,在24孔盤中接種50,000個細胞/孔。至於培養於25ml瓶中的細胞,將五分之一至三分之一的收集量接種於25ml瓶中,以達繼代目的。使用本規程,自下表6-1中所列舉的捐贈者,分離人類RPE。 Human eyes obtained in a wet room were rinsed in PBS with 4% Pen-Strep (Life Technologies, penicillin-streptomycin, 10,000 U / mL, catalog number 15140122), and the anterior segment was removed at the limbus ( If it has not been removed) to open it. After necropsy and photography, the retina was removed and the posterior pole was cut into small pieces (about 1 cm) and cultured in 4 ml of 2% dispase solution (w / v in DMEM, 25 mM HEPES, 200 U / mL) at 37C. Pen-Strep) (Roche Diagnostic Corp, catalog number 04942078001) for 5 minutes. At least two volumes of DMEM (Life Technologies, catalog number 12430054), 25 mM HEPES (Sigma-Aldrich Corp., catalog number H4034-100G), 200U / mL Pen-Strep were added to stop the culture. Place the tissue in a 100 mm Petri dish with sufficient fresh DMEM, 25 mM HEPES, 2% Pen-Strep, and allow it to stand at 37 ° C for 6 to 12 hours. The tissue was transferred to RPMI medium (Life Technologies, catalog number 22400089) in a Petri dish, and RPE cells were teased off by gently scraping or spraying with a pipette tip. The cells were rinsed twice with HBSS (GIBCO, catalog number 310-4170), resuspended in 0.5 ml 0.1% trypsin (pH 8.0, Life Technologies, catalog number 25300054), and incubated at 37C for 2 minutes. The cell mixture was triturated with Pasteur pipette until the RPE cells were dispersed and 5x excess MEM / 20% FBS (minimum essential medium, Lift Technologies, catalog number 1095098; fetal bovine serum) was added , Life Technologies, catalog number 16000036) to stop enzyme treatment. The cells were centrifuged at 1000 xg, resuspended in an appropriate volume of MEM / 20 (MEM containing 20% FBS), counted, and cultured in an appropriate container (24-well dish, Thomas Scientific, catalog number 6901A11; 25ml bottle, VWR, Catalog number 29185-298). Roughly, 50,000 cells / well were seeded in a 24-well dish. As for the cells cultured in the 25ml bottle, one-fifth to one-third of the collected amount is inoculated into the 25ml bottle for the purpose of passage. Using this protocol, human RPE was isolated from the donors listed in Table 6-1 below.
表6-1.所使用之捐贈者
表6-2顯示關於所使用之捐贈者眼睛的額外資訊。 Table 6-2 shows additional information about the donor eye used.
在分離之後,使用以下規程,使先前的RPE細胞進行繼代。接著,隨後在實例7至13中的研究中,使用此等細胞培養物。 After isolation, the following procedure was used to subculture the previous RPE cells. Next, in the studies in Examples 7 to 13, these cell cultures were used.
將人類RPE細胞培養於25ml瓶或24孔盤中以達到長滿。當收集人類RPE細胞時,除了以24孔形式接種細胞之外,25ml瓶亦進行接種以獲得大量細胞以用於儲備、接種額外24孔、及繼代。當該瓶長滿時,使細胞進行繼代。繼續此過程以進行大約6次繼代左右。繼代之間的天數及倍增次數係取決於有多少個細胞接種於瓶中,但是倍增時間似乎為大約2天。瓶常在大約10天內長滿。 Human RPE cells were cultured in 25ml flasks or 24-well dishes to achieve fullness. When collecting human RPE cells, in addition to seeding cells in a 24-well format, a 25 ml bottle was also seeded to obtain a large number of cells for storage, seeding an additional 24 wells, and subculture. When the flask is full, the cells are passaged. Continue this process to carry out about 6 generations. The number of days between generations and the number of doublings depend on how many cells are seeded in the flask, but the doubling time seems to be about 2 days. Bottles often grow up in about 10 days.
至於繼代,將培養物用PBS潤洗2次,用0.1%胰蛋白酶(pH 8.0,Life Technologies,目錄號25300054)進行胰蛋白酶消化,並進行收集。使細胞繼代至另一個瓶或盤(大致1/5至1/3長滿量),或使至少3×105個細胞再懸浮於0.5至1ml凍存培養基(Life Technologies,目錄號12648010)中,於Mr.FrostyTM Freezing Container中逐漸冷卻,且儲存於液態氮中。為了進行再生,將細胞之冷凍管於37℃水浴中回溫,並將細胞轉移至24孔盤之孔中或60mm培養皿中,且培養直到長滿,然後進行繼代。 As for the subculture, the culture was rinsed twice with PBS, trypsinized with 0.1% trypsin (pH 8.0, Life Technologies, catalog number 25300054), and collected. Passage the cells to another flask or dish (approximately 1/5 to 1/3 full volume), or resuspend at least 3 × 10 5 cells in 0.5 to 1 ml cryopreservation medium (Life Technologies, catalog number 12648010) Medium, gradually cooled in Mr. Frosty TM Freezing Container, and stored in liquid nitrogen. For regeneration, the cryotubes of the cells were warmed in a 37 ° C water bath, and the cells were transferred to the wells of a 24-well plate or 60 mm petri dishes, and cultured until they were overgrown, and then subcultured.
人類眼睛係獲自不同眼庫(Florida Lions Eye Bank,Bascom Palmer Eye Institute,Miami,FL;San Diego Eye Bank,San Diego,CA;National Disease Research Interchange(NDRI),Philadelphia,PA)。特定而言,使用表6-1中捐贈者的人類眼睛。將視網膜分離,用Polytron(5mm均質機)均質化,分層於27%至50%線性蔗糖梯度頂部,並在4℃下於SW41轉子中以38,000rpm離心1小時。收集代表不同大小之均質化ROS粒子的ROS帶(可能至多三條帶,即上帶、中帶、下帶),將其以HBSS稀釋,並於HB-4轉子中以7000rpm離心10分鐘以使ROS成為團塊。將ROS團塊再懸浮於無血清培養基中。添加FITC儲備液(2mg/ml於0.1M碳酸氫鈉中,pH 9.0至9.5)(Sigma-Aldrich Corp.,目錄號F7250-50MG)至最終濃度10μg/ml且在室溫下培養4小時。將經FITC染色之ROS藉由於微量離心機中離心以成為團塊,再懸浮於MEM/20中。用血球計估計其濃度,並記錄其顯微鏡下外觀(應顯示一般球形或桿狀結構)。將「活性」部分儲存於4℃下以供日常使用,並將冷凍部分按原樣冷凍於-20℃下以供儲存。 Human eye systems were obtained from different eye banks (Florida Lions Eye Bank, Bascom Palmer Eye Institute, Miami, FL; San Diego Eye Bank, San Diego, CA; National Disease Research Interchange (NDRI), Philadelphia, PA). Specifically, the human eyes of the donors in Table 6-1 are used. The retina was separated, homogenized with a Polytron (5mm homogenizer), layered on top of a 27% to 50% linear sucrose gradient, and centrifuged at 38,000 rpm in a SW41 rotor at 4 ° C for 1 hour. Collect ROS bands that represent homogenized ROS particles of different sizes (possibly up to three bands, namely upper band, middle band, and lower band), dilute them with HBSS, and centrifuge at 7000 rpm in HB-4 rotor for 10 minutes to make ROS Become clumps. Resuspend the ROS pellet in serum-free medium. FITC stock solution (2 mg / ml in 0.1 M sodium bicarbonate, pH 9.0 to 9.5) (Sigma-Aldrich Corp., catalog number F7250-50MG) was added to a final concentration of 10 μg / ml and incubated at room temperature for 4 hours. FITC-stained ROS were pelleted by centrifugation in a microcentrifuge and resuspended in MEM / 20. Estimate its concentration with a hemocytometer and record its appearance under the microscope (should show a generally spherical or rod-like structure). Store the "active" part at 4 ° C for daily use, and freeze the frozen part as it is at -20 ° C for storage.
亦自6至8週齡的Long Evans大鼠及成年豬(屠宰場)獲得眼睛。使用上述關於人類ROS之過程,自大鼠及豬眼睛分離出視桿細胞外節,並加以分類。 Eyes were also obtained from 6-8 weeks old Long Evans rats and adult pigs (slaughterhouses). Using the process described above for human ROS, the outer segments of rod cells were isolated and classified from the eyes of rats and pigs.
如所討論,實例1至5證明了hUTC會救援吞噬,從而在視網膜變性的RCS大鼠模型中減緩視網膜變性。實例7至13顯示使用藉由此實例中方法所獲得之人類RPE細胞及ROS的類似測試之結果。在某些實例中,使用大鼠及豬ROS以進行比較。 As discussed, Examples 1 to 5 demonstrate that hUTC will rescue phagocytosis, thereby slowing retinal degeneration in the RCS rat model of retinal degeneration. Examples 7 to 13 show the results of similar tests using human RPE cells and ROS obtained by the method in this example. In some examples, rat and pig ROS were used for comparison.
在實例6至13中使用下列縮寫: TSP 血小板反應蛋白 The following abbreviations are used in Examples 6 to 13: TSP thrombospondin
此外,如實例6至13中所使用,ROS及RPE細胞將藉由其捐贈者識別來進行識別。舉例而言,14-06-040 RPE係指分離自捐贈者14-06-040之RPE細胞。此外,如實例6至13中所使用,1’意指初代,2’意指次代,諸如此類。 In addition, as used in Examples 6 to 13, ROS and RPE cells will be identified by their donor identification. For example, 14-06-040 RPE refers to RPE cells isolated from donor 14-06-040. Furthermore, as used in Examples 6 to 13, 1 'means the first generation, 2' means the second generation, and the like.
進行使用經分離之人類RPE及ROS的吞噬檢定。 A phagocytosis test using isolated human RPE and ROS was performed.
將用4%多聚甲醛固定於孔(24孔盤)中之RPE細胞,用PBS充分清洗。將細胞以1% BSA(Sigma-Aldrich目錄號A-7030)+10%新生山羊血清(Life Technologies,目錄號16210-064)+0.1% TritonX-100(Sigma-Aldrich,目錄號T-8787)於PBS中培養0.5至1小時,然後與一級抗體(Pan-keratin C11 Mouse mAb,Cell Signaling,目錄號4545S,以含有1% BSA之PBS進行1:400稀釋)在RT下培養2小時或在4C下培養過夜,清洗,且最後與適當Alexa Green或Red共軛之二級抗體(以PBS進行1:50稀釋,在RT下1小時)(Life Technologies,目錄號A21202)培養。對照組是僅在阻斷後經二級抗體處理。在進行最後清洗之後,將製劑固定於 Vectashield封片液(VWR,目錄號101098-042)中,且在裝備有數位照相機的Photomicroscope III下進行檢查。 RPE cells fixed in wells (24-well plate) with 4% paraformaldehyde were washed thoroughly with PBS. Cells were treated with 1% BSA (Sigma-Aldrich catalog number A-7030) + 10% newborn goat serum (Life Technologies, catalog number 16210-064) + 0.1% TritonX-100 (Sigma-Aldrich, catalog number T-8787) in Incubate in PBS for 0.5 to 1 hour, then incubate with primary antibody (Pan-keratin C11 Mouse mAb, Cell Signaling, catalog number 4545S, 1: 400 dilution with PBS containing 1% BSA) at RT for 2 hours or at 4C Incubate overnight, wash, and finally incubate with appropriate Alexa Green or Red conjugated secondary antibodies (diluted 1:50 in PBS, 1 hour at RT) (Life Technologies, catalog number A21202). The control group was treated with secondary antibodies only after blocking. After the final washing, the preparation was fixed in Vectashield mounting solution (VWR, catalog number 101098-042) and checked under Photomicroscope III equipped with a digital camera.
在檢定之前,將5×104個人類RPE細胞(在實例6中分離)接種在於24孔盤之各孔中的圓形玻璃蓋玻片上,維持於MEM/20中至少6天,隨後維持於MEM+5% FBS中。藉由以FITC-ROS(0.5至5×106)覆蓋培養物且在37℃下培養8至12小時,開始檢定。在培養結束時,將細胞用PBS劇烈清洗以移除未攝入之ROS,且以2%多聚甲醛(Paraformaldehyde,4%於PBS中,用PBS稀釋至2%,在稀釋後1週內使用)(Paraformaldehyde,4%於PBS中,VWR,目錄號AAJ61899-AK)固定。藉由螢光顯微鏡法,使用Zeiss Fluorescence Photomicroscope III,以312x放大率,可視化攝入之ROS。檢查至少10個代表性視野(視野大小,0.021mm2),並計數攝入螢光之ROS。 Prior to the assay, 5 × 10 4 human RPE cells (isolated in Example 6) were seeded on round glass coverslips in each well of a 24-well plate, maintained in MEM / 20 for at least 6 days, and then maintained in MEM + 5% FBS. The assay was started by covering the culture with FITC-ROS (0.5 to 5 × 10 6 ) and incubating at 37 ° C. for 8 to 12 hours. At the end of the culture, the cells were vigorously washed with PBS to remove uningested ROS, and 2% paraformaldehyde (Paraformaldehyde, 4% in PBS, diluted with PBS to 2%, used within 1 week after dilution ) (Paraformaldehyde, 4% in PBS, VWR, catalog number AAJ61899-AK) fixed. Using fluorescence microscopy, Zeiss Fluorescence Photomicroscope III was used to visualize the ingested ROS at 312x magnification. Check at least 10 representative visual fields (field size, 0.021 mm 2 ), and count the ROS absorbed into the fluorescent light.
為了證明經培養人類RPE吞噬大鼠ROS的能力,使用2種人類(14-06-040)(1’,第13天)培養物及2種N大鼠RPE(6/4/14、6/13/14)培養物以對大鼠ROS(7.5×106,6/18/14)進行吞噬檢定(8h)。免疫螢光顯微照片(參見圖18)顯示人類細胞吞 噬的證據,然而自體螢光有礙於進行良好定量評估。(自體螢光可能來自於RPE中之脂褐質。) To demonstrate the ability of cultured human RPE to engulf rat ROS, two human (14-06-040) (1 ', day 13) cultures and two N rat RPE (6/4/14, 6 / 13/14) Culture to perform phagocytosis test on rat ROS (7.5 × 10 6 , 6/18/14) (8 h). Immunofluorescence micrographs (see Figure 18) show evidence of human cell phagocytosis, however autofluorescence hinders good quantitative evaluation. (Autofluorescence may come from lipofuscin in RPE.)
先前顯示,大鼠RPE細胞於體外吞噬ROS之時程依循不同的模式,其中結合所添加之ROS至該等細胞,且呈相對靜止直到大約8h為止,此時發生劇烈攝入。在15至16小時,吞噬之第一循環即將結束,而開始進行具類似結合、等待、及攝入之第二循環。為了瞭解是否人類RPE出現相同模式的吞噬,在使用大鼠ROS的情況下,檢查人類RPE及大鼠RPE的吞噬時程。使用如上述之相同細胞。使用大鼠ROS(6/18/14、6/30/14)之混合製劑。將大鼠ROS添加至所培養之細胞,且在第1、2、3、6、8、9、10、11、12、13、15、及16小時停止吞噬並加以分析。在大鼠RPE中出現預期的吞噬時程,亦即,持續1至6h結合但呈低吞噬活性,在8h開始爆發吞噬,持續直到15h,並在16h減緩速度且有下一個循環的證據(參見圖19)。在人類RPE中出現基本上相同的模式,其證實大鼠RPE與人類RPE之間吞噬特徵的相似性。 Previously, it was shown that the phagocytosis of ROS by rat RPE cells in vitro followed a different pattern, in which the added ROS was bound to these cells and remained relatively stationary until about 8 hours, at which time violent uptake occurred. In 15 to 16 hours, the first cycle of phagocytosis is about to end, and a second cycle of similar binding, waiting, and ingestion begins. To understand whether human RPE exhibits the same pattern of phagocytosis, in the case of using rat ROS, examine the phagocytosis time course of human RPE and rat RPE. Use the same cells as above. A mixed preparation of rat ROS (6/18/14, 6/30/14) was used. Rat ROS was added to the cultured cells, and phagocytosis was stopped and analyzed at 1, 2, 3, 6, 8, 9, 10, 11, 12, 13, 15, and 16 hours. The expected phagocytosis time course appeared in rat RPE, ie, it continued to bind for 1 to 6 hours but exhibited low phagocytic activity. Phagocytosis started at 8 hours, continued until 15 hours, and slowed down at 16 hours with evidence of the next cycle (see also Figure 19). A substantially identical pattern appears in human RPE, which confirms the similarity in phagocytic characteristics between rat RPE and human RPE.
用大鼠ROS的另一種製劑,重複吞噬時程實驗。使用人類RPE(14-06-040)(1’,第28天)、大鼠N RPE(7/3/14、7/11/14)、及大鼠ROS(2×106,7/10/14)之培養物。將ROS添加至細胞, 且在第6、8、10、11、13、15、及16h停止吞噬以用於分析。已觀察到如先前實驗所觀察到的相同模式,其證實大鼠RPE與人類RPE吞噬之間的相似性。執行吞噬之部分定量(參見表7-1)。 Using another preparation of rat ROS, the phagocytosis time course experiment was repeated. Use human RPE (14-06-040) (1 ', day 28), rat N RPE (7/3/14, 7/11/14), and rat ROS (2 × 10 6 , 7/10 / 14). ROS was added to the cells, and phagocytosis was stopped at 6, 8, 10, 11, 13, 15, and 16h for analysis. The same pattern as observed in previous experiments has been observed, which confirms the similarity between rat RPE and human RPE phagocytosis. Perform partial phagocytosis (see Table 7-1).
使用兩種大鼠RPE(8-14-14、9-9-14)培養物,以在使用人類ROS製劑(5×106)的情況下測試8小時之吞噬。此測試結果顯示於圖20。藉由大鼠RPE已觀察到吞噬的證據。上帶ROS代表小於中帶及下帶材料的均質化ROS粒子,且似乎顯示較佳吞噬。 Two rat RPE (8-14-14, 9-9-14) cultures were used to test phagocytosis for 8 hours using human ROS preparation (5 × 10 6 ). The test results are shown in Figure 20. Evidence of phagocytosis has been observed by rat RPE. The upper band ROS represents homogenized ROS particles smaller than the middle and lower band materials, and appears to show better phagocytosis.
人類ND04760 RPE之培養物(第11天)係用於進行對上帶及下帶人類ROS(自ND04760視網膜製得,9-18-14)及大鼠ROS(9-25-14)之8h吞噬。吞噬人類上帶及下帶ROS的效果皆較吞噬大鼠ROS的效果為佳。人類ROS吞噬之定量顯示如下(參見表7-2)。 The culture of human ND04760 RPE (day 11) was used for 8h phagocytosis of upper and lower human ROS (made from ND04760 retina, 9-18-14) and rat ROS (9-25-14) . The effect of engulfing human upper and lower ROS is better than that of ROS in rats. The quantification of human ROS phagocytosis is shown below (see Table 7-2).
表7-2.人類ROS吞噬之定量
上帶及下帶之按原樣或隨凍存培養基冷凍的ND04760人類ROS(9/18/14)(各自為10×106)經過測試,測試係針對於24孔培養物中人類RPE(15-02-032)之8h吞噬效率。所有冷凍ROS樣本皆出現有效率的吞噬。此等樣本已經儲存大約6個月。人類ROS吞噬之定量顯示如下(參見表7-3)。 The ND04760 human ROS (9/18/14) (10 × 10 6 each) frozen on the top and bottom bands as they were or frozen with cryopreservation medium were tested. The test is for human RPE (15- 02-032) 8h phagocytosis efficiency. All frozen ROS samples were effectively phagocytosed. These samples have been stored for about 6 months. The quantification of human ROS phagocytosis is shown below (see Table 7-3).
使用15-02-032人類RPE及N大鼠RPE(1/23/15)培養物以進行8h之對人類ROS(15-02-032,2/13/15,各自為5×106)及大鼠ROS(1/27/15,各自為10×106)的吞噬檢定。兩者細胞皆 對大鼠ROS出現低吞噬水準,但是兩者皆對人類ROS出現良好吞噬,因此其證實人類ROS相較於先前所觀察之大鼠ROS的更高效率。針對人類15-02-032結果執行定量(參見表7-4)。 Use 15-02-032 human RPE and N rat RPE (1/23/15) cultures for 8h of human ROS (15-02-032, 2/13/15, 5 × 10 6 each ) and Phagocytosis test of rat ROS (1/27/15, each 10 × 10 6 ). Both cells showed low phagocytic levels for rat ROS, but both showed good phagocytosis of human ROS, so it confirmed that human ROS is more efficient than previously observed rat ROS. Quantification was performed on human 15-02-032 results (see Table 7-4).
將培養物中的15-02-032人類RPE(第24天)以Pan-keratin(C11)單株抗體(Cell Signaling)(1:400)進行免疫染色,以確認其為RPE細胞(參見圖21)。二級抗體係Alexa 488共軛抗小鼠IgG抗體。對照組未經一級抗體處理。獲得RPE細胞的良好染色,而對照組沒有染色,其證實該細胞型為RPE。 15-02-032 human RPE (day 24) in culture was immunostained with Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1: 400) to confirm that it was RPE cells (see Figure 21 ). Secondary Antibody System Alexa 488 conjugated anti-mouse IgG antibody. The control group was not treated with primary antibody. Good staining of RPE cells was obtained, while the control group was not stained, which confirmed that the cell type was RPE.
使於24孔盤中的人類15-03-027 RPE培養物(1’第12天)及大鼠N RPE培養物(2/15/15),進行對大鼠ROS(1/27/15,各自為10×106)及人類ROS(15-03-027,3/12/15,各自為5×106)的8小時吞噬檢定。註:1’意指初代,2’意指次代,諸如此類。以 上皆觀察到良好吞噬。人類RPE及大鼠RPE兩者對人類ROS的吞噬水準皆高於對大鼠ROS。測試結果顯示於下表7-5。 Human 15-03-027 RPE cultures (1 'day 12) and rat N RPE cultures (2/15/15) in a 24-well dish were subjected to rat ROS (1/27/15, 8 × 10 6 phagocytosis test for 10 × 10 6 each ) and human ROS (15-03-027, 3/12/15, 5 × 10 6 each ). Note: 1 'means the first generation, 2' means the second generation, and so on. Good phagocytosis was observed above. Both human RPE and rat RPE have higher levels of phagocytosis of human ROS than rat ROS. The test results are shown in Table 7-5 below.
在第14天收集多個孔的初代15-03-027 RPE培養物,使其繼代至孔中並加以觀察。2’細胞生長良好,並在第15天已長滿,且準備用於實驗中。在細胞已達到長滿之後繼續進行其繼代,並使繼代進行至5’繼代階段。 The primary 15-03-027 RPE culture of multiple wells was collected on day 14 and subcultured into the wells and observed. The 2 ' cells grew well and had grown up on the 15th day, and were ready to be used in experiments. After the cell has reached fullness, proceed to its sub-generation and proceed to the 5 'sub-generation stage.
為了測試經繼代人類RPE之吞噬的能力、及不同ROS在吞噬中的效率,人類RPE 15-03-027之20天3’培養物係用於對大鼠(4/24/15,各自為5×106)、人類(15-03-027,3/12/15,各自為 5×106;及15-04-001,4/3/15,各自為5×106)、及豬(3/26/15,各自為20×106)之ROS進行8h吞噬。此測試結果顯示於表7-6。 In order to test the ability of phagocytosis of subcultured human RPE and the efficiency of different ROS in phagocytosis, the 20-day 3 'culture of human RPE 15-03-027 was used to treat rats (4/24/15 5 × 10 6 ), humans (15-03-027, 3/12/15, each 5 × 10 6 ; and 15-04-001, 4/3/15, each 5 × 10 6 ), and pigs (3/26/15, each 20 × 10 6 ) ROS were phagocytosed for 8 h. The test results are shown in Table 7-6.
所有樣本皆出現良好吞噬;除了人類ROS(15-04-001)的結果表現不如預期。經繼代之3’人類RPE所出現的吞噬水準係與先前類似檢定中之初代RPE所出現的吞噬水準在相同範圍內。不同ROS的相對吞噬效率。結果亦與之前出現的相同。再次,人類ROS顯示相較於大鼠ROS及豬ROS更高的吞噬水準。在不同ROS製劑以及初代與繼代人類RPE之吞噬水準方面,已觀察到一致的模式。 All samples showed good phagocytosis; except for human ROS (15-04-001), the results did not perform as expected. The phagocytosis level of the 3 'human RPE after successive generations is within the same range as the phagocytosis level of the first generation RPE in the previous similar tests. The relative phagocytosis efficiency of different ROS. The result is the same as before. Again, human ROS showed higher phagocytosis levels than rat ROS and porcine ROS. A consistent pattern has been observed with regard to the phagocytosis levels of different ROS preparations and the primary and secondary human RPE.
為了測試經繼代人類RPE之吞噬的能力、及不同ROS在吞噬中的效率,人類RPE 15-03-027之21天2’培養物係用於對大 鼠(4/24/15,各自為7×106)、人類(15-03-027,3/12/15,各自為5×106;及15-04-001,4/3/15,各自為5×106)、及豬(3/26/15,各自為20×106)之ROS進行8h吞噬。測試結果顯示於表7-7。 In order to test the ability of phagocytosis of subcultured human RPE and the efficiency of different ROS in phagocytosis, the 21-day 2 'culture of human RPE 15-03-027 was used to treat rats (4/24/15, each 7 × 10 6 ), humans (15-03-027, 3/12/15, each 5 × 10 6 ; and 15-04-001, 4/3/15, each 5 × 10 6 ), and pigs (3/26/15, each 20 × 10 6 ) ROS were phagocytosed for 8 h. The test results are shown in Table 7-7.
所有樣本皆觀察到良好吞噬,且結果非常類似於實例5中3’15-03-027細胞的結果。因此,針對2’人類15-03-027 RPE培養物觀察到的吞噬水準再次與針對3’及初代人類RPE觀察到的吞噬水準在相同範圍內,而且大鼠、人類、及豬之ROS在吞噬中的相對效率再次如之前所出現的;亦即,人類ROS係優於鼠及豬之ROS。在人類RPE吞噬中,豬ROS顯示最低的效率。因此,在初代對經繼代之人類RPE及不同ROS製劑的吞噬效率方面,再次觀察到一致性。初代與經繼代之人類RPE之間的吞噬水準似乎變化不大。人類ROS似乎為人類RPE的最佳受質。 Good phagocytosis was observed in all samples, and the results were very similar to the results of 3'15-03-027 cells in Example 5. Therefore, the phagocytic level observed for the 2 'human 15-03-027 RPE culture was again within the same range as the phagocytic level observed for the 3' and primary human RPE, and the ROS of rats, humans, and pigs were phagocytic The relative efficiencies in Xia are again as before; that is, human ROS are superior to mouse and pig ROS. Porcine ROS showed the lowest efficiency in human RPE phagocytosis. Therefore, consistency was again observed in the phagocytosis efficiency of the first generation of human RPE and different ROS preparations. The level of phagocytosis between the first generation and subsequent generations of human RPE does not seem to change much. Human ROS seems to be the best substrate for human RPE.
為了測試經繼代人類RPE之吞噬的能力、及不同ROS在吞噬中的效率,人類RPE 15-03-027之40天3’培養物係用於對大鼠(4/24/15,各自為7×106)、人類(15-03-027,3/12/15,各自為5×106;及15-04-001,4/3/15,各自為5×106)、及豬(3/26/15,各自為20×106)之ROS進行8h吞噬。測試結果顯示於表7-8。 In order to test the ability of phagocytosis of subcultured human RPE and the efficiency of different ROS in phagocytosis, the 40-day 3 'culture of human RPE 15-03-027 was used to treat rats (4/24/15, respectively 7 × 10 6 ), humans (15-03-027, 3/12/15, each 5 × 10 6 ; and 15-04-001, 4/3/15, each 5 × 10 6 ), and pigs (3/26/15, each 20 × 10 6 ) ROS were phagocytosed for 8 h. The test results are shown in Table 7-8.
結果類似於20天3’15-03-027人類RPE所獲結果,除了針對大鼠ROS之吞噬水準較高。然而,其維持了3種不同類型ROS之基本相對關係,其中就人類15-03-027 RPE吞噬而言,人類ROS相較於大鼠ROS及豬ROS仍為最佳。豬ROS再次為最差。此等3’RPE之吞噬水準亦與先前3’細胞在相同範圍內。 The results are similar to those obtained at 20 days 3'15-03-027 human RPE, except that the level of phagocytosis against rat ROS is higher. However, it maintains the basic relative relationship of 3 different types of ROS, of which human ROS is still the best compared to rat ROS and porcine ROS in terms of human 15-03-027 RPE phagocytosis. Pig ROS is again the worst. The phagocytic level of these 3'RPEs is also in the same range as the previous 3 'cells.
人類RPE(2’、3’,15-03-027)之冷凍。收集多個孔的經繼代15-03-027人類RPE細胞(在第23天為2’,在第42天為3’,各自大致為106),並將其再懸浮於1ml凍存培養基中。將樣本分為兩份,逐漸降溫,並儲存於液態氮中。稍後測試此等經冷凍儲存細胞之存活性及功能性。 Freezing of human RPE ( 2 ' , 3' , 15-03-027 ). Collect multiple wells of passaged 15-03-027 human RPE cells (2 'on day 23, 3' on day 42, approximately 10 6 each ), and resuspend in 1 ml of cryopreservation medium in. Divide the sample into two, gradually cool down, and store in liquid nitrogen. The viability and functionality of these frozen storage cells will be tested later.
冷凍人類RPE(7/22/15)之再生。將自5/21/15冷凍且儲存之2’及3’人類15-03-027 RPE細胞之部分解凍,並使其再生長。細胞再生大致需要3週。 Regeneration of frozen human RPE (7/22/15) . Part of 2 'and 3' human 15-03-027 RPE cells frozen and stored from 5/21/15 were thawed and allowed to re-grow. Cell regeneration takes approximately 3 weeks.
為了測試冷凍人類ROS製劑用於吞噬之能力,將各種ROS製劑的冷凍樣本解凍,並用於吞噬檢定。所測試的冷凍ROS製劑包括:1.NDRI #1 04760 ROS(9/18/14),3/10/15再冷凍;2.15-02-032人類ROS(2/13/15),3/6/15冷凍;3.15-03-027人類ROS(3/12/15),3/24/15冷凍;4.15-04-001人類ROS(4/3/15),4/27/15冷凍。在解凍後,各自使用5×106個ROS,供15-03-027 3’(第103天)人類RPE培養物進行8小時吞噬。測試結果顯示於表7-9。 To test the ability of frozen human ROS preparations for phagocytosis, frozen samples of various ROS preparations were thawed and used for phagocytosis assays. The frozen ROS preparations tested include: 1.NDRI # 1 04760 ROS (9/18/14), re-frozen on 3/10/15; 2.15-02-032 human ROS (2/13/15), 3/6 / 15 frozen; 3.15-03-027 human ROS (3/12/15), 3/24/15 frozen; 4.15-04-001 human ROS (4/3/15), 4/27/15 frozen. After thawing, 5 × 10 6 ROS were used each for phagocytosis of 15-03-027 3 ′ (day 103) human RPE cultures for 8 hours. The test results are shown in Table 7-9.
所有經冷凍與解凍的人類ROS製劑皆出現良好吞噬水準,其在預期範圍內。因此,經冷凍與解凍的人類ROS製劑對於吞噬實驗來說,似乎完全可以接受。 All frozen and thawed human ROS preparations showed good phagocytosis levels, which were within the expected range. Therefore, frozen and thawed human ROS preparations seem completely acceptable for phagocytosis experiments.
為了確認15-04-001人類RPE之吞噬的能力,且為了測試人類RPE吞噬大鼠、人類、及豬之ROS的效率,15-04-001人類RPE之17天培養物係用於對各為5×106個的大鼠ROS(4/24/15)、15-03-027人類ROS(3/12/15)、15-04-001人類ROS(4/3/15)、及20×106個豬ROS(3/26/15)的8h吞噬檢定。亦對初代15-04-001人類RPE的第32天培養物及初代15-04-001人類RPE的第55天培養物執行本實驗。測試結果顯示於表7-10(17天培養物)、表7-11(32天培養物)、及表7-12(55天培養物)。 To confirm the ability of 15-04-001 human RPE to engulf, and to test the efficiency of human RPE to engulf ROS in rats, humans, and pigs, 17-04-001 human RPE 17-day cultures were used for 5 × 10 6 rat ROS (4/24/15), 15-03-027 human ROS (3/12/15), 15-04-001 human ROS (4/3/15), and 20 × 10 6 swine ROS (3/26/15) 8h phagocytosis test. This experiment was also carried out on the day 32 culture of the primary 15-04-001 human RPE and the day 55 culture of the primary 15-04-001 human RPE. The test results are shown in Table 7-10 (17-day culture), Table 7-11 (32-day culture), and Table 7-12 (55-day culture).
17天培養物之結果。觀察到預期的吞噬模式。對3種不同種類ROS之吞噬水準係與之前所觀察到的在相同範圍內。再 次,人類ROS顯示最佳吞噬,而大鼠ROS為人類之大約50%,且豬ROS顯示最低活性。因此,維持了一致性。 Results of the 17- day culture. The expected phagocytosis pattern was observed. The phagocytic levels for the three different types of ROS are in the same range as previously observed. Again, human ROS showed the best phagocytosis, while rat ROS was about 50% of human, and porcine ROS showed the lowest activity. Therefore, consistency is maintained.
32天培養物之結果。對15-04-001人類ROS之吞噬並未作用。其餘樣本顯示良好吞噬,而且在大鼠、人類、及豬之ROS的吞噬水準範圍及相對效率方面,其預期吞噬模式如之前所顯示。在所出現的吞噬模式方面,初代人類RPE的第17天及第32天培養物之間差異似乎不大。 The results of the 32- day culture. It has no effect on 15-04-001 phagocytosis of human ROS. The remaining samples showed good phagocytosis, and the expected phagocytosis pattern was as previously shown in terms of the phagocytic level range and relative efficiency of ROS in rats, humans, and pigs. In terms of the phagocytosis patterns that appeared, the differences between the cultures of day 17 and day 32 of the primary human RPE did not seem to differ much.
55天培養物之結果。獲得類似於15-04-001之第17天及第32天初代RPE培養物的結果,其證實在吞噬水準、不同ROS製劑之相對效率、及細胞不同培養期之相等性方面的一致模式。 Results of the 55- day culture. Results similar to the primary RPE cultures on days 17 and 32 of 15-04-001 were obtained, which confirmed a consistent pattern in terms of phagocytosis level, relative efficiency of different ROS preparations, and equivalence of cells in different culture periods.
為了檢查不同繼代之人類RPE的吞噬效率,利用次代15-04-001人類RPE之第30天培養物重複上述實驗(參見下表7-13)。用次代培養物所獲得的吞噬模式基本上與15-04-001之初代RPE培養物所出現的相同,其證實在吞噬水準及不同ROS製劑之相 對效率方面的一致模式。15-04-001細胞展示出人類RPE培養物之不同繼代的吞噬相等性。 In order to check the phagocytosis efficiency of different generations of human RPE, the above experiment was repeated using the 30th day culture of the next generation 15-04-001 human RPE (see Table 7-13 below). The phagocytosis pattern obtained with the secondary culture was basically the same as that seen in the primary RPE culture of 15-04-001, which confirmed a consistent pattern in terms of phagocytosis level and the relative efficiency of different ROS preparations. 15-04-001 cells exhibited equal phagocytosis of different passages of human RPE culture.
為了測試冷凍人類ROS製劑用於吞噬之能力,將各種人類ROS製劑的冷凍樣本解凍,並用於吞噬檢定。所測試的冷凍ROS製劑包括:1.NDRI #1 04760 ROS(9/18/14),3/10/15再冷凍;2.15-02-032人類ROS(2/13/15),3/6/15冷凍;3.15-03-027人類ROS(3/12/15),3/24/15冷凍;4.15-04-001人類ROS(4/3/15),4/27/15冷凍。在解凍後,各自使用5×106個ROS,供15-04-001 2’(第85天)人類RPE培養物進行8小時吞噬。亦使用3’15-03-027人類RPE執行相同實驗。測試結果顯示於表7-14。 To test the ability of frozen human ROS preparations for phagocytosis, frozen samples of various human ROS preparations were thawed and used for phagocytosis assays. The frozen ROS preparations tested include: 1.NDRI # 1 04760 ROS (9/18/14), re-frozen on 3/10/15; 2.15-02-032 human ROS (2/13/15), 3/6 / 15 frozen; 3.15-03-027 human ROS (3/12/15), 3/24/15 frozen; 4.15-04-001 human ROS (4/3/15), 4/27/15 frozen. After thawing, 5 × 10 6 ROS were used each for phagocytosis of 15-04-001 2 ′ (day 85) human RPE cultures for 8 hours. The same experiment was also performed using 3'15-03-027 human RPE. The test results are shown in Table 7-14.
所有經冷凍與解凍的人類ROS製劑皆出現良好吞噬水準。15-02-032及15-03-027 ROS之吞噬水準皆高於平常,這可能是因為此等ROS製劑顯示小於平常的粒子,導致細胞中更多可見的攝入ROS粒子。如3’15-03-027細胞所示,經冷凍與解凍的ROS製劑對於吞噬實驗來說,似乎完全可以接受。 All frozen and thawed human ROS preparations showed good phagocytosis. 15-02-032 and 15-03-027 The phagocytosis level of ROS is higher than usual, which may be because these ROS preparations show smaller particles than usual, leading to more visible uptake of ROS particles in the cells. As shown by 3'15-03-027 cells, frozen and thawed ROS preparations seem to be completely acceptable for phagocytosis experiments.
為了檢查人類ROS不同製劑(蔗糖梯度離心中之不同帶)之吞噬效率,15-11-098 ROS之上帶、中帶、及下帶係針對3’第44天及4’第42天15-04-001 RPE之吞噬經過測試。測試結果顯示於表7-15。 In order to check the phagocytosis efficiency of different preparations of human ROS (different bands in sucrose gradient centrifugation), 15-11-098 ROS upper band, middle band, and lower band are for 3 'day 44 and 4' day 42 15- 04-001 RPE phagocytosis has been tested. The test results are shown in Table 7-15.
用中帶ROS所獲得之吞噬水準大致上在平常範圍內。然而,用上帶ROS所獲得之吞噬水準係高於其他者。再次,原因可能是上帶中富含較小ROS粒子;其造成濃度低估,這是因為彼等在亮視野中較難以可視化,而且在螢光下細胞內有更多可見粒子。下帶所獲得之水準有些多變,且其他RPE培養物的結果對此作出證明,如下所示。總吞噬水準的變化性可能存在,其取決於ROS粒子的性質,但是可藉由使用跨樣本的相同ROS製劑而獲得一致性。 The level of phagocytosis obtained with mid-band ROS is roughly within the normal range. However, the level of phagocytosis obtained with top band ROS is higher than others. Again, the reason may be that the upper band is rich in smaller ROS particles; it causes the concentration to be underestimated because they are more difficult to visualize in bright fields and there are more visible particles in the cells under fluorescence. The level obtained in the lower band is somewhat variable, and the results of other RPE cultures demonstrate this, as shown below. The variability of the total phagocytosis level may exist, depending on the nature of the ROS particles, but consistency can be obtained by using the same ROS preparation across samples.
為了測試可能受污染之人類RPE經不同類型清洗對吞噬之影響,將15-07-072初代RPE培養物(懷疑可能有細菌污染)用Betadine清洗3分鐘或用含有2x Pen-Strep之PBS清洗3分鐘,並使其進行對各為5×106個的上帶或下帶15-07-072 ROS(7/28/15)之8h吞噬。此測試結果顯示於表7-16(上帶)或表7-17(下帶)。 To test the effect of different types of cleaning on human phagocytosis of human RPE that may be contaminated, 15-07-072 primary RPE cultures (suspicion of possible bacterial contamination) were washed with Betadine for 3 minutes or with PBS containing 2x Pen-Strep 5 minutes, and allowed to phagocytose 15-07-072 ROS (7/28/15) for 8x each of 5 × 10 6 upper or lower bands. The test results are shown in Table 7-16 (upper band) or Table 7-17 (lower band).
亦進行此實驗之二重複。此測試結果顯示於表7-18(上帶及下帶(第2個實驗))。 The second repeat of this experiment was also conducted. The results of this test are shown in Table 7-18 (upper belt and lower belt (second experiment)).
用Betadine清洗對細胞似乎過於嚴峻,而用含有2x P-S之PBS清洗並未傷害細胞,且細胞顯示預期吞噬水準。2x P-S清洗看來亦會消除培養物中的細菌污染。 Washing with Betadine seemed too severe for the cells, while washing with PBS containing 2x P-S did not harm the cells, and the cells showed the expected level of phagocytosis. 2x P-S cleaning also seems to eliminate bacterial contamination in the culture.
表7-17.不同清洗劑對吞噬之作用(下帶)
為了比較人類2’及3’RPE(後者濃度為50k或小於50k)之吞噬水準,使15-07-072 2’及3’(每培養物50k及小於50k)RPE培養物進行對各為2.5×106個的15-07-072上帶ROS之8小時吞噬檢定。此測試結果顯示於表7-19。 To compare the phagocytic levels of human 2 'and 3' RPE (the latter concentration is 50k or less), the 15-07-072 2 'and 3' (50k and less than 50k per culture) RPE cultures were paired to 2.5 × 10 6 8-hour phagocytosis tests with ROS on 15-07-072. The test results are shown in Table 7-19.
全部3個樣本皆觀察到預期的吞噬水準,其證明2’及3’培養物的相等性,且亦證明培養物之濃度為50k及小於50k的相等性。 The expected level of phagocytosis was observed in all 3 samples, which proved the equality of the 2 'and 3' cultures, and also proved the equality of the cultures with a concentration of 50k and less than 50k.
為了檢查人類ROS不同製劑(蔗糖梯度離心中之不同帶)之吞噬效率,15-11-098 ROS之上帶、中帶、及下帶係針對5’ 第62天及6’第24天15-07-072 RPE之吞噬經過測試。此測試結果顯示於表7-20。 In order to check the phagocytosis efficiency of different preparations of human ROS (different bands in sucrose gradient centrifugation), the 15-11-098 ROS upper, middle, and lower bands were targeted at 5 'day 62 and 6' day 24 07-072 RPE phagocytosis has been tested. The test results are shown in Table 7-20.
如之前所出現的,用中帶ROS所獲得之吞噬水準大致上在平常範圍內。用上帶ROS所獲得之吞噬水準係再次高於其他者。(再次,原因最有可能是因為上帶中富含較小ROS粒子。)在使用15-07-072細胞的情況下,此次用下帶所獲得之吞噬水準係高於預期,其反映如之前所提及之此帶的變化性。 As previously shown, the level of phagocytosis obtained with mid-band ROS is roughly within the normal range. The level of phagocytosis obtained with top band ROS is again higher than the others. (Again, the reason is most likely because the upper zone is rich in smaller ROS particles.) In the case of using 15-07-072 cells, the phagocytosis level obtained with the lower zone is higher than expected, which reflects The variability of this band mentioned earlier.
此時SD1 AMD RPE之初代培養物正快速惡化,因此執行最後一種吞噬檢定,該檢定係比較2種正常人類初代RPE培養物(15-09-027,1’,第41天;15-08-074,1’,第62天)與San Diego 1「乾性」AMD初代RPE培養物(L,1’,第48天;R,1’,第48天)對各為5×106個的人類ROS(15-10-021,上帶)之8小時吞噬水準。此測試結果顯示於表7-21。 At this time, the primary culture of SD1 AMD RPE is rapidly deteriorating, so the final phagocytosis test is performed, which compares two normal human primary RPE cultures (15-09-027, 1 ', day 41; 15-08- 074, 1 ', day 62) with San Diego 1 "dry" AMD primary RPE cultures (L, 1', day 48; R, 1 ', day 48) for 5 × 10 6 humans each The phagocytosis level of ROS (15-10-021, top) is 8 hours. The test results are shown in Table 7-21.
觀察到SD1 AMD初代RPE相較於正常人類初代RPE之吞噬水準的相同降低,其證實AMD RPE之吞噬水準降低。R與L AMD RPE兩者顯示出相較於此時2種正常細胞(15-09-027及15-08-074)的降低,且如之前所出現的,所降低之水準係在60百分位數。 The same reduction in the phagocytic level of the SD1 AMD primary RPE compared to normal human primary RPE was observed, which confirmed that the AMD RPE phagocytic level was reduced. Both R and L AMD RPE showed a decrease compared to the two normal cells (15-09-027 and 15-08-074) at this time, and as previously appeared, the level of reduction was 60% Digits.
將培養物中的SD1 AMD L 5’RPE(第69天)以Pan-keratin(C11)單株抗體(Cell Signaling)(1:400)進行免疫染色,以確認此等AMD細胞為RPE細胞。二級抗體係Alexa 488共軛抗小鼠IgG抗體。對照組未經一級抗體處理。獲得RPE細胞的良好染色,而對照組沒有染色,其證實該細胞型為RPE(參見圖22)。 The SD1 AMD L 5 'RPE (day 69) in the culture was immunostained with Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1: 400) to confirm that these AMD cells were RPE cells. Secondary Antibody System Alexa 488 conjugated anti-mouse IgG antibody. The control group was not treated with primary antibody. Good staining of RPE cells was obtained, while the control group was not stained, which confirmed that the cell type was RPE (see FIG. 22).
將培養物中的4’15-10-021濕性AMD RPE(第56天)以Pan-keratin(C11)單株抗體(Cell Signaling)(1:400)進行免疫染色,以確認此等AMD細胞為RPE細胞。二級抗體係Alexa 488共軛抗小鼠IgG抗體。對照組未經一級抗體處理。獲得RPE細胞的良好染色,而對照組沒有染色,其證實該細胞型為RPE(參見圖23)。 The 4'15-10-021 wet AMD RPE (day 56) in the culture was immunostained with Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1: 400) to confirm these AMD cells RPE cells. Secondary Antibody System Alexa 488 conjugated anti-mouse IgG antibody. The control group was not treated with primary antibody. Good staining of RPE cells was obtained, while the control group was not stained, which confirmed that the cell type was RPE (see FIG. 23).
將培養物中的2’ND08333「AMD」RPE(第15天)以Pan-keratin(C11)單株抗體(Cell Signaling)(1:400)進行免疫染色,以確認此等AMD細胞為RPE細胞。二級抗體係Alexa 488共軛抗小鼠IgG抗體。對照組未經一級抗體處理。獲得RPE細胞的良好染色,而對照組沒有染色,其證實該細胞型為RPE(參見圖24)。 The 2'ND08333 "AMD" RPE (day 15) in the culture was immunostained with Pan-keratin (C11) monoclonal antibody (Cell Signaling) (1: 400) to confirm that these AMD cells were RPE cells. Secondary Antibody System Alexa 488 conjugated anti-mouse IgG antibody. The control group was not treated with primary antibody. Good staining of RPE cells was obtained, while the control group was not stained, which confirmed that the cell type was RPE (see FIG. 24).
為了檢查人類ROS不同製劑(蔗糖梯度離心中之不同帶)之吞噬效率,15-11-098 ROS之上帶、中帶、及下帶係針對4’第51天及5’第31天之15-08-074 RPE培養物之吞噬經過測試。如之前所出現的,用中帶ROS所獲得之吞噬水準大致上在平常範圍內。用上帶ROS所獲得之吞噬水準係再次高於其他者。在使用15-08-074細胞的情況下,此次用下帶所獲得之吞噬水準係高於預期,其反映如之前所提及之此帶的變化性。因此,總吞噬水準的變化性可能存在,其取決於ROS粒子的性質,但是可藉由使用跨樣本的相同ROS製劑而獲得一致性。此測試結果顯示於下表7-22。 In order to check the phagocytosis efficiency of different preparations of human ROS (different bands in sucrose gradient centrifugation), 15-11-098 ROS upper band, middle band, and lower band are for 15 days of 4 'day 51 and 5' day 31 -08-074 The phagocytosis of RPE culture was tested. As previously shown, the level of phagocytosis obtained with mid-band ROS is roughly within the normal range. The level of phagocytosis obtained with top band ROS is again higher than the others. In the case of using 15-08-074 cells, the phagocytic level obtained with this lower band is higher than expected, which reflects the variability of this band as mentioned previously. Therefore, variability in the total phagocytosis level may exist, depending on the nature of the ROS particles, but consistency can be obtained by using the same ROS preparation across samples. The test results are shown in Table 7-22 below.
獲得實例6中分離出之數種人類RPE的RNA水準。特定言之,使用以下規程測定此等RNA水準。 The RNA levels of several human RPEs isolated in Example 6 were obtained. Specifically, these RNA levels are determined using the following procedure.
將於24孔盤中之RPE培養物用PBS清洗3x。將1ml Trizol(Life Technologies,目錄號15596026)添加至細胞,並根據製造商的規程分離RNA。根據製造商的規程,用DNAFree(Applied Biosystems,目錄號AM1906)自製劑移除污染的基因組DNA。用BioRad分光光度計或Nanodrop 2000,測定RNA製劑之濃度。 RPE cultures in 24-well dishes will be washed 3x with PBS. 1 ml Trizol (Life Technologies, catalog number 15596026) was added to the cells, and RNA was isolated according to the manufacturer's protocol. According to the manufacturer's protocol, DNAFree (Applied Biosystems, catalog number AM1906) was used to remove contaminated genomic DNA from the preparation. Using BioRad spectrophotometer or Nanodrop 2000, determine the concentration of RNA preparation.
自2孔的15-03-027 RPE培養物(第13天)分離RNA。 RNA was isolated from a 2-well 15-03-027 RPE culture (day 13).
1. 0.15μg/μl,260/280=3.04,18μl 1. 0.15μg / μl, 260/280 = 3.04,18μl
2. 0.08μg/μl,260/280=1.94,18μl 2. 0.08μg / μl, 260/280 = 1.94,18μl
自3孔的第24天15-04-001初代人類RPE培養物(於24孔盤中)分離RNA。 RNA was isolated from the 3-well day 24-04-001 primary human RPE culture (in a 24-well dish).
結果: Results:
0.05μg/μl,260/280=2.50,18μl 0.05μg / μl, 260/280 = 2.50,18μl
0.18μg/μl,260/280=4.21,18μl 0.18μg / μl, 260/280 = 4.21,18μl
0.13μg/μl,260/280=1.58,18μl 0.13μg / μl, 260/280 = 1.58,18μl
自15-04-001人類RPE之初代培養物(1’,第217天)分離RNA。 RNA was isolated from the primary culture of 15-04-001 human RPE (1 ', day 217).
結果: Results:
0.04μg/μl,260/280=2.48,18μl 0.04μg / μl, 260/280 = 2.48,18μl
V.自初代15-07-072 RPE分離RNA V. RNA isolation from the first generation 15-07-072 RPE
自2個15-07-072 RPE培養物(在第16天)樣本分離RNA。 RNA was isolated from two 15-07-072 RPE culture (at day 16) samples.
結果: Results:
1. 0.2μg/μl,260/280=1.45,18μl 1. 0.2μg / μl, 260/280 = 1.45,18μl
2. 0.2μg/μl,260/280=1.89,18μl 2. 0.2μg / μl, 260/280 = 1.89,18μl
自濕性AMD 15-10-021 RPE之第75天初代培養物分離RNA。 RNA was isolated from the primary culture on day 75 of the wet AMD 15-10-021 RPE.
結果:1’,第75天,0.06μg/μl,260/280=1.80,18μl Results: 1 ’, Day 75, 0.06μg / μl, 260/280 = 1.80,18μl
自兩個以下樣本分離RNA:(a)1’第26天ND08626 AMD RPE培養物;及(b)1’第26天ND08626 AMD RPE培養物。 RNA was isolated from two of the following samples: (a) 1 'day 26 ND08626 AMD RPE culture; and (b) 1' day 26 ND08626 AMD RPE culture.
結果: Results:
1’第16天,0.04μg/μl,260/280=1.92,18μl 1 ’Day 16, 0.04μg / μl, 260/280 = 1.92,18μl
1’第16天,0.03μg/μl,260/280=2.00,18μl 1’Day 16, 0.03μg / μl, 260/280 = 2.00,18μl
1’第26天,0.05μg/μl,260/280=2.14,18μl 1 ’Day 26, 0.05μg / μl, 260/280 = 2.14, 18μl
1’第26天,0.08μg/μl,260/280=1.89,18μl Day 26 on 1 ’, 0.08μg / μl, 260/280 = 1.89,18μl
自2孔的15-08-074 1’第17天RPE培養物分離RNA。 RNA was isolated from 2-well 15-08-074 1 'day 17 RPE culture.
結果: Results:
1. 0.17μg/μl,260/280=1.78,18μl 1. 0.17μg / μl, 260/280 = 1.78,18μl
2. 0.15μg/μl,260/280=2.64,18μl 2. 0.15μg / μl, 260/280 = 2.64,18μl
為了比較來自不同年齡個體之RPE的吞噬水準,使人類眼睛15-07-072(來自39歲捐贈者)之5’(第200天)、15-04-001(來自59歲捐贈者)之5’(第63天)、15-08-074(來自61歲捐贈者)之5’(第48天)、15-11-098(71歲)之4’(第20天)、及15-09-027(來自79歲捐贈者)之5’(第82天)的正常RPE進行對來自人類眼睛15-11-098之上帶、中帶、及下帶ROS製劑(12/2/15,各自為5×105)之8小時吞噬。大致上,在24孔盤中接種50,000個細胞/孔。為進行額外比較,來自稍早自人類眼睛15-02-032(來自31歲捐贈者)分離之RPE的吞噬實驗之結果亦包括在內。(註:5’意指第5繼代)此測試結果顯示於圖25A及圖25B。數據顯示人類RPE的吞噬功能隨著年齡(尤其是在60歲之後)而下降。 In order to compare the phagocytosis level of RPE from individuals of different ages, the human eye was 5 '(day 200) of 15-07-072 (from a 39-year-old donor), and 5 of 15-04-001 (from a 59-year-old donor) '(Day 63), 15-08-074 (from a 61-year-old donor) 5' (Day 48), 15-11-098 (71-year-old) 4 '(Day 20), and 15-09 -027 (from a 79-year-old donor) 5 '(day 82) of normal RPE was performed on the upper, middle, and lower ROS preparations from the human eye 15-11-098 (12/2/15, each It is 5 × 10 5 ) for 8 hours. Roughly, 50,000 cells / well were seeded in a 24-well dish. For additional comparison, the results of phagocytosis experiments from RPE isolated earlier from human eye 15-02-032 (from a 31-year-old donor) were also included. (Note: 5 'means the 5th generation) The test results are shown in Figure 25A and Figure 25B. The data shows that the phagocytic function of human RPE decreases with age (especially after age 60).
為了比較AMD與正常RPE之吞噬水準,使San Diego 1 AMD左4’第20天和右3’第11天、15-10-021「濕性」AMD 3’第18天、15-09-027 N 3’第31天和4’第19天、15-08-074 N 3’第41天 和4’第34天、15-07-072 N 5’第45天、及15-04-001 N 1’第209天、3’第27天、4’第25天之RPE進行對各為2×106個的15-10-021上帶ROS之8小時吞噬檢定。如從圖26可得知,所有AMD RPE之吞噬水準顯著地低於正常RPE之吞噬水準。(15-04-001 1’之吞噬並未作用。此可能歸因於培養物中的細胞狀態,因為該等細胞已經達到衰老且停止進行吞噬。) To compare the phagocytic levels of AMD and normal RPE, San Diego 1 AMD left 4 'day 20 and right 3' day 11, 15-10-021 "wet" AMD 3 'day 18, 15-09-027 N 3 'Day 31 and 4' Day 19, 15-08-074 N 3 'Day 41 and 4' Day 34, 15-07-072 N 5 'Day 45, and 15-04-001 N 8 hours phagocytosis test was carried out on 15-10-021 with 2 × 106 RPEs at 1 ′ day 209, 3 ′ day 27, and 4 ′ day 25. As can be seen from FIG. 26, the phagocytic level of all AMD RPEs is significantly lower than that of normal RPE. (The phagocytosis of 15-04-001 1 ’has no effect. This may be due to the state of the cells in the culture, because these cells have reached senescence and stop phagocytosis.)
為了比較AMD ND08333與正常RPE之吞噬水準,使ND08333 AMD 3’第42天、15-09-027正常5’第96天、及15-08-074正常5’第62天之RPE進行對各為5×105個的15-11-098上帶ROS之8小時吞噬檢定。AMD ND08333 RPE之吞噬水準係遠低於正常(15-09-027及15-08-074)RPE之吞噬水準,其再次證實所觀察到的模式。 In order to compare the phagocytic levels of AMD ND08333 and normal RPE, the RPE of ND08333 AMD 3 'day 42, 15-09-027 normal 5' day 96, and 15-08-074 normal 5 'day 62 5 × 10 5 8-11 hours phagocytosis test with ROS on 15-11-098. The phagocytosis level of AMD ND08333 RPE is much lower than the normal (15-09-027 and 15-08-074) RPE phagocytosis level, which again confirms the observed pattern.
為了再次比較AMD ND08333與正常RPE之吞噬水準,使ND08333 AMD 3’第43天、15-04-001正常5’第78天、及15-07-072正常6’第115天之RPE進行對各為5×105個的15-11-098上帶ROS之8小時吞噬檢定。AMD ND08333相較於正常(15-04-001及15-07-072)RPE所減少的吞噬水準再次經過確認。 In order to compare the phagocytic levels of AMD ND08333 and normal RPE again, the RPE of ND08333 AMD 3 'day 43, 15-04-001 normal 5' day 78, and 15-07-072 normal 6 'day 115 were compared. 5 × 105 8-hour phagocytosis test with ROS on 15-11-098. The reduced phagocytosis level of AMD ND08333 compared to normal (15-04-001 and 15-07-072) RPE has been confirmed again.
為了比較AMD ND08626與正常RPE之吞噬水準,使ND08626 AMD 1’第17天、15-09-027 N 5’第88天、及15-08-074 N 5’第55天之RPE進行對各為5×105個的15-11-098上帶ROS之8小時吞噬檢定。AMD ND08626 RPE之吞噬水準顯著地低於正常RPE(15-09-027及15-08-074)之吞噬水準,其證實一致觀察到的模式。 In order to compare the phagocytosis level of AMD ND08626 and normal RPE, the RPE of ND08626 AMD 1 'day 17, 15-09-027 N 5' day 88, and 15-08-074 N 5 'day 55 were compared 5 × 10 5 8-11 hours phagocytosis test with ROS on 15-11-098. The phagocytosis level of AMD ND08626 RPE is significantly lower than that of normal RPE (15-09-027 and 15-08-074), which confirms the pattern observed consistently.
為了再次比較AMD ND08626與正常RPE之吞噬水準,使ND08626 AMD 1’第17天、及15-11-098 N 3’第40天和4’第24天之RPE進行對各為5×105個的15-11-098上帶ROS之8小時吞噬檢定。AMD ND08626 RPE之吞噬水準係遠低於正常15-11-098 RPE之吞噬水準,其再次證實一致觀察到的模式。 In order to compare the phagocytosis levels of AMD ND08626 and normal RPE again, the RPE of ND08626 AMD 1 'day 17 and 15-11-098 N 3' day 40 and 4 'day 24 were 5 × 10 5 each 8-hour phagocytosis test with ROS on 15-11-098. The phagocytosis level of AMD ND08626 RPE is much lower than the normal phagocytosis level of 15-11-098 RPE, which again confirms the pattern observed consistently.
為了再次比較AMD ND08626與正常RPE之吞噬水準,使ND08626 AMD 1’第24天、15-04-001 N 5’第77天、及15-07-072 N 6’第123天之RPE進行對各為5×105個的15-11-098上帶ROS之8小時吞噬檢定。AMD ND08626 RPE之吞噬水準係遠低於正常15-04-001及15-07-072 RPE之吞噬水準,其再次證實一致觀察到的模式。 In order to compare the phagocytosis levels of AMD ND08626 and normal RPE again, the RPE of ND08626 AMD 1 'day 24, 15-04-001 N 5' day 77, and 15-07-072 N 6 'day 123 were compared It is an 8-hour phagocytosis test with 5 × 10 5 15-11-098 with ROS. The phagocytosis level of AMD ND08626 RPE is much lower than the normal phagocytosis levels of 15-04-001 and 15-07-072 RPE, which again confirms the pattern observed consistently.
為了再次比較AMD ND08626與正常RPE之吞噬水準,使ND08626 AMD 1’第25天、及15-08-074 N 4’第111天和6’第82天之RPE進行對各為5×105個的15-11-098上帶ROS之8小時吞噬檢定。AMD ND08626 RPE之吞噬水準再次遠低於正常15-08-074 RPE之吞噬水準,其再次證實一致觀察到的模式。 In order to compare the phagocytosis level of AMD ND08626 and normal RPE again, the RPE of ND08626 AMD 1 'day 25, and 15-08-074 N 4' day 111 and 6 'day 82 were 5 × 10 5 each 8-hour phagocytosis test with ROS on 15-11-098. The phagocytosis level of AMD ND08626 RPE is again much lower than the normal 15-08-074 RPE phagocytosis level, which again confirms the pattern observed consistently.
使各兩孔的人類15-08-074 1’第24天RPE培養物進行對各為5×106個的15-08-074上帶及下帶ROS製劑之8小時吞噬檢定。觀察到吞噬,但是水準偏低;可能歸因於非常小的ROS粒子,其難以可視化及計數,導致漏計。 Two-well human 15-08-074 1 'day 24 RPE cultures were subjected to an 8-hour phagocytosis test for 5 × 10 6 each of 15-08-074 upper and lower band ROS preparations. Phagocytosis is observed, but the level is low; it may be due to very small ROS particles, which are difficult to visualize and count, resulting in missed counts.
為了測試15-08-074上帶及下帶ROS製劑(8/25/15)之吞噬效率,使15-03-027 5’冷凍/再生、15-07-072 3’第43天、15-04-001 2’第134天和4’冷凍/再生、及15-08-074 1’第15天之RPE培養物進行對ROS製劑之吞噬。觀察到吞噬,但是水準皆偏低。(非常小的ROS粒子難以可視化及計數。) In order to test the phagocytosis efficiency of 15-08-074 upper and lower band ROS preparations (8/25/15), 15-03-027 5 'freezing / regeneration, 15-07-072 3' day 43, 15- 04-001 2 'Day 134 and 4' Freeze / Regeneration, and 15-08-074 1 'Day 15 RPE cultures were phagocytosed for ROS preparations. Engulfing was observed, but the level was low. (Very small ROS particles are difficult to visualize and count.)
為了比較不同繼代之人類RPE培養物的吞噬水準,使15-08-074 2’第46天、3’第20天、及4’第13天之RPE進行對各為2×106個的15-10-021人類上帶ROS之吞噬。在不同繼代之RPE培養物觀察到相等吞噬水準,其證實不同繼代之人類RPE培養物的相等性。 In order to compare the phagocytosis levels of different generations of human RPE cultures, 15-08-074 2 ′ day 46, 3 ′ day 20, and 4 ′ day 13 RPE were performed for 2 × 10 6 15-10-021 Engulfed with ROS on humans. Equal phagocytic levels were observed in RPE cultures of different passages, which confirmed the equality of human RPE cultures of different passages.
為了比較AMD RPE(San Diego 1 L濕性(不再有R)4’第21天;15-10-021濕性3’第19天)與正常RPE(15-09-027 3’第32天、4’第20天;15-08-074 3'第42天、4'第35天;15-07-072 5'第46天;15-04-001 1’第210天、3’第28天、4’第26天)之吞噬水準,使RPE培養物各自對2×106個15-09-027上帶ROS進行8h吞噬檢定、固定、及分析。如之前所出現的,AMD RPE之吞噬水準係低於全部正常RPE之吞噬水準。 To compare AMD RPE (San Diego 1 L wet (no longer R) 4 'day 21; 15-10-021 wet 3' day 19) with normal RPE (15-09-027 3 'day 32 , 4 '20th day; 15-08-074 3' 42nd day, 4 '35th day; 15-07-072 5' 46th day; 15-04-001 1 '210th day, 3' 28th Day, 4 'Day 26) phagocytosis level, RPE cultures were used for 8h phagocytosis test, fixation and analysis of 2 × 10 6 ROS on 15-09-027. As previously shown, the phagocytosis level of AMD RPE is lower than that of all normal RPE.
為了比較正常(15-11-098 1’第48天、2’第12天)與AMD(San Diego 1 L 5’第60天;15-10-021濕性5’第40天)RPE(經及未經CM11處理24小時)之吞噬水準,使RPE培養物因此各自對1×106個15-11-098培養基ROS進行8h吞噬檢定、固定、及分析。 In order to compare normal (15-11-098 1 'day 48, 2' day 12) and AMD (San Diego 1 L 5 'day 60; 15-10-021 wet 5' day 40) RPE (after And the phagocytosis level without CM11 treatment for 24 hours), so that the RPE cultures were each subjected to 8x phagocytosis assay, fixation, and analysis on 1 × 10 6 ROS of 15-11-098 medium.
兩種AMD RPE(SD1 L、15-10-021濕性)之吞噬水準皆遠低於正常RPE(15-11-098)之吞噬水準。CM11處理基本上使AMD RPE所降低之吞噬水準正常化。兩者結果確認了之前觀察到的情形。 The phagocytic levels of the two AMD RPEs (SD1 L, 15-10-021 wetness) are much lower than the phagocytic levels of normal RPE (15-11-098). CM11 treatment basically normalized the reduced phagocytosis level of AMD RPE. The results of both confirm the previously observed situation.
為了判定ROS最佳量以用於15-11-098 ROS製劑(上帶、中帶、下帶)之吞噬檢定,使15-08-074 5’第43天之RPE培養物進行對各為1×105、5×105、及1×106個的上帶、中帶、及下帶15-11-098 ROS製劑之吞噬檢定。絕對吞噬水準反映上述結果,亦即,上帶及下帶為偏高,中帶為較低。觀察到對於不同量之各別ROS的預期劑量反應,其指示5×105個可係此ROS製劑之適合用量。劑量反應測試結果顯示於圖27。 In order to determine the optimal amount of ROS to be used for the phagocytosis test of 15-11-098 ROS preparations (upper band, middle band, lower band), 15-08-074 5 'day 43 RPE cultures were tested for 1 15-15-098 ROS preparation phagocytosis test for × 10 5 , 5 × 10 5 , and 1 × 10 6 upper, middle, and lower bands. The absolute phagocytosis level reflects the above results, that is, the upper and lower bands are higher, and the middle band is lower. The expected dose response to different amounts of individual ROS was observed, which indicated that 5 × 10 5 could be a suitable dosage for this ROS preparation. The results of the dose response test are shown in Figure 27.
為了判定ROS最佳量以用於15-11-098 ROS製劑(上帶、中帶、下帶)之吞噬檢定,使15-11-098 4’第14天之RPE培養物進行對各為1×105、5×105、及1×106個的上帶、中帶、及下帶15-11-098 ROS製劑之吞噬檢定。絕對吞噬水準反映上述15-09-027結果,亦即,上帶及下帶為偏高,中帶為較低。觀察到對於不同量之各別ROS的預期劑量反應,其指示5×105可係此ROS製劑(U,L)之適合用量。劑量反應測試結果顯示於圖28。 In order to determine the optimal amount of ROS for the phagocytosis test of the 15-11-098 ROS preparation (upper band, middle band, lower band), the 15-11-098 4 'day 14 RPE cultures were tested for 1 15-15-098 ROS preparation phagocytosis test for × 10 5 , 5 × 10 5 , and 1 × 10 6 upper, middle, and lower bands. The absolute level of phagocytosis reflects the above results of 15-09-027, that is, the upper and lower bands are higher, and the middle band is lower. The expected dose response for different amounts of individual ROS was observed, indicating that 5 × 10 5 may be a suitable dosage for this ROS preparation (U, L). The results of the dose response test are shown in Figure 28.
為了檢查人類ROS不同製劑(蔗糖梯度離心中之不同帶)之吞噬效率,15-11-098 ROS之上帶、中帶、及下帶係針對3’第48天、4’第36天、及5’第7天15-09-027 RPE培養物之吞噬經 過測試。測試結果顯示於表9-1。如之前所出現的,用中帶ROS所獲得之吞噬水準大致上在平常範圍內。用上帶ROS所獲得之吞噬水準係再次高於其他者。(再次,原因最有可能是因為上帶中富含較小ROS粒子。)在使用15-09-027細胞的情況下,此次用下帶所獲得之吞噬水準係高於預期,其反映如之前所提及之此帶的變化性。總吞噬水準的變化性可能存在,其取決於ROS粒子的性質,但是可藉由使用跨樣本的相同ROS製劑而獲得一致性。 In order to check the phagocytosis efficiency of different preparations of human ROS (different bands in sucrose gradient centrifugation), the upper, middle, and lower bands of 15-11-098 ROS were directed at 3'day 48, 4'day 36, and 5 'Day 7 15-09-027 RPE cultures were tested for phagocytosis. The test results are shown in Table 9-1. As previously shown, the level of phagocytosis obtained with mid-band ROS is roughly within the normal range. The level of phagocytosis obtained with top band ROS is again higher than the others. (Again, the reason is most likely because the upper band is rich in smaller ROS particles.) In the case of using 15-09-027 cells, the phagocytosis level obtained with the lower band was higher than expected, which reflects The variability of this band mentioned earlier. The variability of the total phagocytosis level may exist, depending on the nature of the ROS particles, but consistency can be obtained by using the same ROS preparation across samples.
即使是在相較於年齡最匹配之正常RPE時,此實例中來自AMD及非AMD患者之RPE的各種吞噬檢定顯示AMD RPE有減少的吞噬。 Even when compared to normal age-matched normal RPE, various phagocytosis tests from RPE from AMD and non-AMD patients in this example showed reduced phagocytosis of AMD RPE.
已評估hUTC條件培養基添加至AMD RPE對吞噬之作用。 The effect of hUTC conditioned medium added to AMD RPE on phagocytosis has been evaluated.
將hUTC CM 5(第5製劑:CM5 10k及CM5 11k)、CM 10(第10製劑)、CM11(第11製劑)、CM13(第13製劑)、CM14及CM15(CM14及CM15係於一種製劑中製備)用於實驗。使用條件培養基及對照培養基。為製備CM5,在第1天將hUTC以10,000個存活細胞/cm2(針對CM5 10k)或11,000個存活細胞/cm2(針對CM5 11k)接種於T225細胞培養瓶中之hUTC生長培養基(DMEM低葡萄糖+15% FBS+4mM L-麩醯胺酸)中。為製備CM10、CM11、CM13、CM14、及CM15,在第1天將hUTC以10,000個存活細胞/cm2接種於T75細胞培養瓶中之hUTC生長培養基中。細胞接種後,將其於37℃下、5% CO2培養器中培養24小時。在第2天,吸出培養基,並在CM5之情況下補充63mL/T225瓶之DMEM/F12完全培養基(DMEM:F12培養基+10% FBS+Pen(50 U/ml)/Strep(50μg/ml)),在CM10、CM11之情況下補充20mL/T75瓶之DMEM/F12完全培養基,且在CM13之情況下補充21mL/T75瓶之DMEM/F12完全培養基。再培養細胞48小時。亦將單獨對照培養基(DMEM:F12培養基+10% FBS+Pen(50U/ml)/Strep(50μg/ml))培養48h。在第4天,收集細胞培養物上清液及對照培養基,並將其在4℃下以250g離心5min,然後以1mL/管等分於1.8mL凍存管中,且立即冷凍於-70℃冰箱。 HUTC CM 5 (5th preparation: CM5 10k and CM5 11k), CM 10 (10th preparation), CM11 (11th preparation), CM13 (13th preparation), CM14 and CM15 (CM14 and CM15 in one preparation (Preparation) for experiment. Use conditioned medium and control medium. To prepare CM5, hUTC was inoculated into hUTC growth medium (DMEM low) in T225 cell culture flasks at 10,000 viable cells / cm 2 (for CM5 10k) or 11,000 viable cells / cm 2 (for CM5 11k) on day 1. Glucose + 15% FBS + 4mM L-glutamic acid). To prepare CM10, CM11, CM13, CM14, and CM15, hUTC was inoculated into hUTC growth medium in T75 cell culture flasks at 10,000 viable cells / cm 2 on day 1. After the cells were seeded, they were cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. On the second day, aspirate the medium and supplement with 63mL / T225 bottles of DMEM / F12 complete medium (DMEM: F12 medium + 10% FBS + Pen (50 U / ml) / Strep (50 μg / ml)) , In the case of CM10, CM11 supplemented with 20mL / T75 bottles of DMEM / F12 complete medium, and in the case of CM13 supplemented with 21mL / T75 bottles of DMEM / F12 complete medium. The cells were incubated for another 48 hours. A separate control medium (DMEM: F12 medium + 10% FBS + Pen (50U / ml) / Strep (50 μg / ml)) was also cultured for 48h. On the fourth day, the cell culture supernatant and control medium were collected and centrifuged at 250g for 5 min at 4 ° C, then aliquoted in 1.8mL cryovials at 1mL / tube, and immediately frozen at -70 ° C refrigerator.
為了比較正常人類初代RPE與AMD(「乾性」)初代RPE(經及未經CM處理)之吞噬能力,使San Diego #1「乾性AMD」初代RPE培養物(L,第19天;R,第19天)、正常人類15-09-027初代RPE培養物(第21天)、及正常人類15-08-074初代RPE培養物(第42天)(經及未經CM10處理過夜)進行對各為5×106個的人類15-09-027 ROS之8小時吞噬檢定。測試結果顯示於下表10-1。 To compare the phagocytic capacity of normal human primary RPE and AMD ("dry") primary RPE (with and without CM treatment), San Diego # 1 "dry AMD" primary RPE culture (L, day 19; R, day 19 days), normal human 15-09-027 primary RPE culture (day 21), and normal human 15-08-074 primary RPE culture (day 42) (overnight with and without CM10 treatment) 8 hours phagocytosis test for 5 × 10 6 human 15-09-027 ROS. The test results are shown in Table 10-1 below.
L與R AMD RPE兩者(尤其是R)皆顯示相較於正常(15-09-027)更低的吞噬水準,而且CM10處理增加其等全部(包括正常RPE)之吞噬水準。15-08-074正常者之吞噬並未作用。整體而言,15-08-074之攝入程度異常地低,其指示此RPE培養物無法進行吞噬。 Both L and R AMD RPE (particularly R) showed lower phagocytic levels than normal (15-09-027), and CM10 treatment increased the phagocytic levels of all others (including normal RPE). 15-08-074 The phagocytosis of normal subjects has no effect. Overall, the intake level of 15-08-074 is abnormally low, which indicates that this RPE culture cannot be phagocytosed.
執行實驗以檢定正常人類初代RPE(15-09-027,1’,第29天)及San Diego #1「乾性」AMD初代RPE(L,1’,第27天;R,1’,第27天)(經及未經CM10處理24小時)之吞噬,並亦自各樣本分離RNA。進行吞噬檢定時,使用各為5×106個的人類ROS(15-09-027,上帶)。 Perform experiments to characterize normal human primary RPE (15-09-027, 1 ', day 29) and San Diego # 1 "dry" AMD primary RPE (L, 1', day 27; R, 1 ', day 27 Days) (with and without CM10 treatment for 24 hours), and RNA was also isolated from each sample. For phagocytosis, 5 × 10 6 human ROS (15-09-027, top tape) were used.
RPE培養物在實驗期間剝落,因此破壞了吞噬檢定。然而,仍然自其中一些樣本分離出RNA,如下所示:正常15-09-027 RPE,0.18μg/μl,260/280=?,18μl The RPE culture flakes during the experiment, thus destroying the phagocytosis assay. However, RNA was still isolated from some of the samples as follows: normal 15-09-027 RPE, 0.18 μg / μl, 260/280 =? , 18μl
正常15-09-027 RPE,經CM10處理24h,0.15μg/μl,260/280=6.59,18μl Normal 15-09-027 RPE, treated with CM10 for 24h, 0.15μg / μl, 260/280 = 6.59, 18μl
SD1「乾性」AMD RPE,L,0.1μg/μl,260/280=?,18μl SD1 "dry" AMD RPE, L, 0.1 μg / μl, 260/280 =? , 18μl
SD1「乾性」AMD RPE,L,經CM10處理24h,0.1μg/μl,260/280=?,18μl SD1 "dry" AMD RPE, L, treated with CM10 for 24h, 0.1μg / μl, 260/280 =? , 18μl
一些在260/280測量後的問號指示BioRad分光光度計在測量此等樣本時故障。 Some question marks after the 260/280 measurement indicate that the BioRad spectrophotometer failed when measuring these samples.
基本上,執行與上述相同的實驗,除了所有初代培養物大1天,且CM處理為6小時。由於沒有足夠的SD1 R RPE細胞之初代培養物,因此並未自此等細胞分離RNA。 Basically, the same experiment as above was performed, except that all primary cultures were 1 day old, and the CM treatment was 6 hours. Since there were not enough primary cultures of SD1 R RPE cells, RNA was not isolated from these cells.
此等樣本遭受如先前實驗中的相同局面,亦即培養物剝落,因此毀壞了吞噬檢定。儘管如此,仍對此等樣本進行RNA分離。結果如下:正常15-09-027 RPE,0.08μg/μl,260/280=1.80,18μl These samples suffered the same situation as in the previous experiment, that is, the flaking of the culture, thus ruining the phagocytosis test. Nonetheless, RNA isolation of these samples is still performed. The results are as follows: normal 15-09-027 RPE, 0.08μg / μl, 260/280 = 1.80, 18μl
正常15-09-027 RPE,經CM10處理6h,0.3μg/μl,260/280=1.61,18μl Normal 15-09-027 RPE, treated with CM10 for 6h, 0.3μg / μl, 260/280 = 1.61,18μl
SD1「乾性」AMD RPE,L,0.18μg/μl,260/280=2.38,18μl SD1 "Dry" AMD RPE, L, 0.18μg / μl, 260/280 = 2.38, 18μl
SD1「乾性」AMD RPE,L,經CM10處理6h,0.1μg/μl,260/280=1.47,18μl SD1 "dry" AMD RPE, L, treated with CM10 for 6h, 0.1μg / μl, 260/280 = 1.47,18μl
為了比較濕性AMD與正常之人類RPE(經及未經CM處理24小時)的吞噬水準,且為了自濕性AMD細胞分離RNA,使初代14天「濕性」AMD 15-10-021 RPE、初代37天正常15-09-027 RPE、及次代35天和三代9天正常15-08-074 RPE(經及未經CM10添加)進行對各為5×106個的15-09-027上帶ROS之8小時吞噬檢定。亦自「濕性」AMD 15-10-021 RPE(亦經及未經CM10添加)分離RNA。吞噬檢定之結果顯示於表10-2。 To compare the phagocytic level of wet AMD with normal human RPE (with and without CM treatment for 24 hours), and to isolate RNA from wet AMD cells, the first 14 days of "wet" AMD 15-10-021 RPE, The first generation 37 days normal 15-09-027 RPE, and the second generation 35 days and third generation 9 days normal 15-08-074 RPE (with and without CM10 addition) were performed on 15-09-027 of 5 × 10 6 each 8 hours phagocytosis test with ROS. RNA was also isolated from "wet" AMD 15-10-021 RPE (also added with and without CM10). The results of the phagocytosis test are shown in Table 10-2.
許多樣本在實驗期間經歷剝落,以致僅有「濕性」AMD 15-10-021(經及未經CM)及正常15-08-074 3’(未經CM添加)存活而能夠進行分析。然而,此等結果顯示AMD RPE再次相較於正常者呈較低的吞噬水準(如San Diego「乾性」AMD RPE所示)、以及其經添加CM10而「回復」至正常水準。 Many samples experienced flaking during the experiment, so that only the "wet" AMD 15-10-021 (with and without CM) and normal 15-08-074 3 '(without CM addition) survived and could be analyzed. However, these results show that AMD RPE once again showed a lower phagocytosis level than normal (as shown by San Diego's "dry" AMD RPE), and that it was "reverted" to normal levels by adding CM10.
自15-10-021濕性AMD RPE(經及未經CM10處理24小時)分離之RNA如下:未經CM10處理者,0.08μg/μl,260/280=1.69,18μl RNA isolated from 15-10-021 wet AMD RPE (with and without CM10 treatment for 24 hours) is as follows: those without CM10 treatment, 0.08 μg / μl, 260/280 = 1.69, 18 μl
經CM10處理24小時者,0.02μg/μl,260/280=0.36,18μl CM10 treated for 24 hours, 0.02μg / μl, 260/280 = 0.36,18μl
為了比較「濕性」AMD RPE與正常RPE之吞噬水準,使1’第19天「濕性」AMD 15-10-021 RPE(經及未經CM10處理6小時)、及正常15-08-074 1’第63天、2’第40天、及3’第14天RPE培養物進行對各為2×106個的15-10-021 ROS之8h吞噬檢定。亦自經及未經CM10處理之AMD RPE分離RNA。吞噬檢定之結果顯示於表10-3。 In order to compare the phagocytosis level of "wet" AMD RPE and normal RPE, the "wet" AMD 15-10-021 RPE (with and without CM10 treatment for 6 hours) on day 19 of 1 'and normal 15-08-074 RPE cultures of 1'day 63, 2'day 40, and 3'day 14 were subjected to an 8h phagocytosis test for 15-10-021 ROS of 2 × 10 6 each. RNA was also isolated from AMD RPE with and without CM10 treatment. The results of the phagocytosis test are shown in Table 10-3.
如之前所觀察到的,在1’、2’、及3’15-08-074 RPE中觀察到大致相等的吞噬水準,其證實不同繼代RPE培養物之相等性。絕對計數係非常高,其可能歸因於主要由非常小ROS粒子所組成之15-10-021 ROS製劑。(小ROS粒子似乎導致過度吞噬及較高變化性,這是因為難以可視化小ROS粒子。)可能歸因於此,在此 實驗中,「濕性」AMD 15-10-021 RPE之吞噬水準僅略低於正常者,其與多次觀察到顯著較低之水準形成對比。將CM10添加至AMD RPE如先前所出現的增加了吞噬水準,而維持已觀察到的模式。 As previously observed, approximately equal phagocytic levels were observed in 1 ', 2', and 3 '15-08-074 RPE, which confirmed the equivalence of different subcultures of RPE. The absolute count is very high, which may be attributed to the 15-10-021 ROS preparation which is mainly composed of very small ROS particles. (Small ROS particles seem to cause excessive phagocytosis and high variability, which is because it is difficult to visualize small ROS particles.) It may be attributed to this. In this experiment, the "wet" AMD 15-10-021 RPE phagocytosis level was only Those slightly lower than normal are in contrast to the significantly lower levels observed many times. Adding CM10 to AMD RPE increased the phagocytosis level as previously appeared, while maintaining the observed pattern.
自經及未經CM10處理6小時之濕性AMD 15-10-021 RPE所分離之RNA:未經CM10處理者,0.2μg/μl,260/280=1.43,18μl RNA isolated from wet AMD 15-10-021 RPE with and without CM10 treatment for 6 hours: 0.2μg / μl, 260/280 = 1.43,18μl without CM10 treatment
經CM10處理6h者,0.08μg/μl,260/280=1.19,18μl After treated by CM10 for 6h, 0.08μg / μl, 260/280 = 1.19,18μl
為了比較AMD與正常(N)RPE之吞噬水準,且為了檢查用hUTC CM處理的效果,使下列之RPE(經及未經CM11處理)進行對數種ROS製劑(在此情況下為15-09-027上帶ROS製劑)之8小時吞噬檢定:San Diego 1 AMD左4’(第21天);15-10-021「濕性」AMD 3’(第19天);15-09-027 N 3’(第19天)和4’(第20天);15-08-074 N 3’(第42天)、4’(第35天)、和5’(第15天);15-07-072 N 5’(第46天);及15-04-001 N 2’(第210天)、3’(第28天)、和4’(第26天)。 In order to compare the phagocytic level of AMD and normal (N) RPE, and to check the effect of treatment with hUTC CM, the following RPE (with and without CM11 treatment) were subjected to several ROS preparations (in this case 15-09- 027 with ROS preparation) 8 hours phagocytosis test: San Diego 1 AMD left 4 '(day 21); 15-10-021 "wet" AMD 3' (day 19); 15-09-027 N 3 '(Day 19) and 4' (Day 20); 15-08-074 N 3 '(Day 42), 4' (Day 35), and 5 '(Day 15); 15-07- 072 N 5 '(day 46); and 15-04-001 N 2' (day 210), 3 '(day 28), and 4' (day 26).
2種AMD RPE與8種正常RPE(15-04-001,4’似乎未作用)在未經CM處理下的吞噬水準比較顯示,自兩名AMD捐贈者的眼睛所分離之RNA的吞噬水準顯著地低於自八名正常捐贈者的眼睛所分離之RNA的平均吞噬水準。此與AMD RPE相較於正常RPE降低吞噬之觀察一致。當將AMD RPE之吞噬水準特別與於年齡最匹配之正常RPE比較時,在15-10-021「濕性」AMD樣本觀察到顯著降低情形,但是未在SD1 AMD左樣本觀察到(參見底部計算)。此測試結果顯示於圖29。 Comparison of the phagocytosis levels of 2 AMD RPEs and 8 normal RPEs (15-04-001, 4 'seems to have no effect) without CM treatment showed that the phagocytosis levels of RNA isolated from the eyes of two AMD donors were significant It is lower than the average level of phagocytosis of RNA isolated from the eyes of eight normal donors. This is consistent with the observation that AMD RPE reduces phagocytosis compared to normal RPE. When comparing the AMD RPE phagocytosis level with the age-matched normal RPE, a significant reduction was observed in the 15-10-021 "wet" AMD sample, but not in the SD1 AMD left sample (see bottom calculation ). The test results are shown in Figure 29.
所有樣本(經及未經CM處理)之吞噬水準比較顯示,經過hUTC CM處理後的吞噬水準有顯著增加。在AMD樣本中,吞噬降低情形超過經過CM處理後而正常化者。甚至在正常樣本中,hUTC CM處理促進其吞噬水準(除了一個樣本(15-07-072,5’))。此亦與CM處理增加人類RPE吞噬之模式一致。 The comparison of the phagocytic levels of all samples (with and without CM treatment) showed that the phagocytic levels after hUTC CM treatment increased significantly. In AMD samples, phagocytosis decreased more than normalized after CM treatment. Even in normal samples, hUTC CM treatment promoted its phagocytic level (except for one sample (15-07-072, 5 ')). This is also consistent with the pattern of CM treatment increasing human RPE phagocytosis.
使用來自另一名捐贈者的RPE細胞,重複先前實驗。為了比較AMD與N RPE之吞噬水準,且為了檢查用hUTC CM處理的效果,使下列之RPE(經及未經CM11處理)進行對數種ROS製劑(在此情況下為15-10-021上帶ROS製劑)之8小時吞噬檢定:San Diego 1 AMD左4’(第21天);15-10-021「濕性」AMD 3’(第19天);15-09-027 N 3’(第19天)和4’(第20天);15-08-074 N 3’(第42天)、4’(第35天)、和5’(第15天);15-07-072 N 5’(第46天);及15-04-001 N 2’(第210天)、3’(第28天)、和4’(第26天)。 Repeat the previous experiment using RPE cells from another donor. In order to compare the phagocytic levels of AMD and N RPE, and to check the effect of treatment with hUTC CM, the following RPE (with and without CM11 treatment) were subjected to several ROS preparations (in this case 15-10-021 on the belt ROS preparation) 8 hours phagocytosis test: San Diego 1 AMD left 4 '(day 21); 15-10-021 "wet" AMD 3' (day 19); 15-09-027 N 3 '(day 19 days) and 4 '(day 20); 15-08-074 N 3' (day 42), 4 '(day 35), and 5' (day 15); 15-07-072 N 5 '(Day 46); and 15-04-001 N 2' (day 210), 3 '(day 28), and 4' (day 26).
獲得與用ROS製劑執行之相同實驗相同的結果。當檢查未經hUTC CM處理的結果時,兩種AMD RPE(即SD1「乾性AMD」L及15-10-021「濕性」AMD)之吞噬水準顯著低於全部9種正常RPE之平均水準。此再次與觀察到的模式一致。當將AMD RPE之吞噬水準特別與年齡最匹配之正常樣本比較時,兩種AMD樣本皆顯示相較於正常樣本顯著降低的吞噬水準。CM11處理使全部RPE(包括AMD RPE)之吞噬水準增加(數據未顯示)。CM11處理後,AMD RPE中減少的吞噬水準基本上經正常化。此再次與觀察到的模式一致。 The same results as the same experiment performed with the ROS preparation were obtained. When examining the results without hUTC CM treatment, the phagocytosis levels of the two AMD RPEs (ie, SD1 "Dry AMD" L and 15-10-021 "Wet" AMD) were significantly lower than the average of all 9 normal RPEs. This is again consistent with the observed pattern. When comparing the AMD RPE phagocytosis level with the age-matched normal samples, both AMD samples showed significantly lower phagocytosis levels compared to the normal samples. CM11 treatment increased the phagocytic level of all RPEs (including AMD RPE) (data not shown). After CM11 treatment, the reduced phagocytosis level in AMD RPE was basically normalized. This is again consistent with the observed pattern.
用來自第三名捐贈者的RPE細胞,重複先前實驗。為了比較AMD與正常(N)RPE之吞噬水準,且為了檢查用CM處理的效果,使下列之RPE(經及未經CM11處理)進行對數種ROS製劑(在此情況下為SD1上帶ROS製劑)之8小時吞噬檢定:San Diego 1 AMD左4’(第21天);15-10-021「濕性」AMD 3’(第19天);15-09-027 N 3’(第19天)和4’(第20天);15-08-074 N 4’(第35天)、和5’(第15天);15-07-072 N 5’(第46天);及15-04-001 N 3’(第28天)。 Repeat the previous experiment with RPE cells from the third donor. In order to compare the phagocytosis level of AMD and normal (N) RPE, and to check the effect of treatment with CM, the following RPE (with and without CM11 treatment) were subjected to several ROS preparations (in this case, SD1 with ROS preparations) ) Of 8 hours phagocytosis test: San Diego 1 AMD left 4 '(day 21); 15-10-021 "wet" AMD 3' (day 19); 15-09-027 N 3 '(day 19) ) And 4 '(day 20); 15-08-074 N 4' (day 35), and 5 '(day 15); 15-07-072 N 5' (day 46); and 15- 04-001 N 3 '(Day 28).
獲得與用其他ROS製備執行之先前實驗相同的結果。當檢查未經CM處理的結果時,兩種AMD RPE(即SD1「乾性AMD」L及15-10-021「濕性」AMD)之吞噬水準顯著低於全部6種正常RPE之平均吞噬水準(數據未顯示)。此再次與觀察到的模式一致。當將AMD RPE之吞噬水準特別與年齡最匹配之正常樣本比較時,兩種AMD樣本相較於正常樣本皆顯著降低吞噬水準。 The same results as previous experiments performed with other ROS preparations were obtained. When examining the results without CM treatment, the phagocytic levels of the two AMD RPEs (ie, SD1 "Dry AMD" L and 15-10-021 "Wet" AMD) were significantly lower than the average phagocytic levels of all six normal RPE ( Data not shown). This is again consistent with the observed pattern. When comparing the AMD RPE phagocytosis level with the age-matched normal samples, both AMD samples significantly reduced the phagocytosis level compared to the normal samples.
CM11處理使全部RPE(包括AMD RPE)之吞噬水準增加(數據未顯示)。CM11處理後,AMD RPE中減少的吞噬水準基本上經正常化。此再次與模式一致。 CM11 treatment increased the phagocytic level of all RPEs (including AMD RPE) (data not shown). After CM11 treatment, the reduced phagocytosis level in AMD RPE was basically normalized. This is again consistent with the pattern.
為了比較AMD(ND08333)與正常(15-11-098)之RPE的吞噬水準(經及未經CM處理),使ND08333 AMD 1’第34天和2’第8天、及正常15-11-098 2’第14天之RPE(經及未經CM11處理)進行對各為2×106個的15-11-098中帶ROS之8小時吞噬檢定。再次,如之前所出現的,相較於正常15-11-098 RPE,AMD ND08333 RPE的吞噬水準係較低。CM11對於所有RPE皆具有刺激作用,然而其對於AMD RPE之作用相較於先前所觀察之其他者似乎不大。 In order to compare the phagocytosis level of RPE between AMD (ND08333) and normal (15-11-098) (with and without CM treatment), ND08333 AMD 1 'day 34 and 2' day 8 and normal 15-11- 098 2 'Day 14 RPE (with and without CM11 treatment) was tested for 8 hours phagocytosis with ROS in 15-11-098 of 2 × 10 6 each. Again, as seen previously, the phagocytosis level of AMD ND08333 RPE is lower than the normal 15-11-098 RPE. CM11 has a stimulating effect on all RPE, but its effect on AMD RPE seems to be less than that previously observed.
為了再次比較AMD(ND08333)與正常(15-11-098)之RPE的吞噬水準(經及未經hUTC CM處理),使ND08333 AMD 2’第19天和3’第6天、及正常15-11-098 2’第25天和3’第11天之RPE(經及未經hUTC CM11處理)進行對各為1×106個的15-11-098上帶ROS之8小時吞噬檢定。AMD ND08333 RPE之吞噬水準顯著低於正常15-11-098 RPE(數據未顯示),其證實已出現的模式。hUTC CM11處理對於所有RPE之吞噬皆具有顯著刺激作用,其使AMD RPE中降低的吞噬水準正常化(數據未顯示)。 In order to compare the phagocytosis level of AMD (ND08333) and normal (15-11-098) RPE again (with and without hUTC CM treatment), ND08333 AMD 2 'day 19 and 3' day 6 and normal 15- 11-098 RPE (with and without hUTC CM11 treatment) on 2 'day 25 and 3' day 11 were tested for 8 hours phagocytosis with ROS on 15-11-098 of 1 × 10 6 each. The phagocytosis level of AMD ND08333 RPE is significantly lower than the normal 15-11-098 RPE (data not shown), which confirms the pattern that has emerged. The hUTC CM11 treatment had a significant stimulating effect on all RPE phagocytosis, which normalized the reduced phagocytic level in AMD RPE (data not shown).
為了比較AMD ND08626與正常之RPE的吞噬水準(經及未經hUTC CM處理),使下列之RPE(經及未經CM8 10k處理)進行對各為5×105個的15-11-098上帶ROS之8小時吞噬檢定:ND08626 AMD 2’第21天和3’第3天;15-11-098 N 3’第60天和4’第47天;15-09-027 N 5’第109天;15-08-074 N 4’第123天和5’第75天;及15-04-001 N 5’第90天。AMD ND08626 2’、3’RPE之吞噬水準遠低於正常RPE(15-11-098 3’、4’;15-09-027 5’;15-08-074 4’、5’;15-04-001 5’)。CM8處理增加所有RPE之吞噬,包括基本上經正常化之AMD RPE。維持了所觀察到的模式。 In order to compare the phagocytosis level of AMD ND08626 and normal RPE (with and without hUTC CM treatment), the following RPE (with and without CM8 10k treatment) were performed on 15-11-098 of 5 × 10 5 each 8-hour phagocytosis test with ROS: ND08626 AMD 2 'day 21 and 3' day 3; 15-11-098 N 3 'day 60 and 4' day 47; 15-09-027 N 5 'day 109 Days; 15-08-074 N 4 'day 123 and 5' day 75; and 15-04-001 N 5 'day 90. The phagocytosis level of AMD ND08626 2 ', 3' RPE is much lower than normal RPE (15-11-098 3 ', 4'; 15-09-027 5 '; 15-08-074 4', 5 '; 15-04 -001 5 '). CM8 treatment increases the phagocytosis of all RPEs, including AMD RPE that is substantially normalized. The observed pattern is maintained.
為了比較AMD(ND08626)RPE與正常RPE(15-11-098、15-09-027、15-08-074、15-04-001)(經及未經CM處理)之吞噬水準,使下列進行對各為5×105個的15-11-098上帶ROS之8h吞噬:ND08626 2’第24天和3’第6天RPE;及15-11-098 N 3’第63天、4'第50天;15-09-027 N 5'第112天;15-08-074 N 4'第126天、5'第78天;及15-04-001 N 5'第93天之RPE培養物(經及未經CM5 10k處理24h)。如之前所出現的,AMD ND08626 RPE之吞噬水準係低於正常15-11-098、15-09-027、15-08-074及15-04-001 RPE。亦如之前所出現的,將AMD ND08626 RPE以CM5 10k處理 會使降低的吞噬水準正常化。將正常RPE以CM處理亦使其全部的吞噬水準刺激至可變程度。 In order to compare the phagocytic levels of AMD (ND08626) RPE and normal RPE (15-11-098, 15-09-027, 15-08-074, 15-04-001) (with and without CM treatment), the following 8h phagocytosis of ROS on 15-11-098 with 5 × 10 5 each: ND08626 2 'Day 24 and 3' Day 6 RPE; and 15-11-098 N 3 'Day 63, 4' Day 50; 15-09-027 N 5 'day 112; 15-08-074 N 4' day 126, 5 'day 78; and 15-04-001 N 5' day 93 RPE culture (With and without CM5 10k treatment for 24h). As previously shown, the phagocytosis level of AMD ND08626 RPE is lower than the normal 15-11-098, 15-09-027, 15-08-074 and 15-04-001 RPE. As also seen before, treatment of AMD ND08626 RPE with CM5 10k will normalize the reduced phagocytosis level. Treatment of normal RPE with CM also stimulated all its phagocytic levels to a variable degree.
此實例中的測試確認CM對於AMD及正常RPE兩者之吞噬皆具有刺激作用,其使AMD RPE之水準基本上正常化。 The test in this example confirmed that CM has a stimulating effect on both the phagocytosis of both AMD and normal RPE, which substantially normalizes the level of AMD RPE.
已評估添加hUTC條件培養基對於RPE RNA水準之作用。 The effect of adding hUTC conditioned medium on RPE RNA levels has been evaluated.
為了檢查CM對於正常人類RPE透過RNA之基因表現的作用,將15-04-001人類RPE(4’,第20天)培養物用CM11處理6及24小時,且自該等培養物以及對照樣本分離RNA。將對照樣本培養於MEM5中6小時。 In order to examine the effect of CM on the gene expression of normal human RPE through RNA, 15-04-001 human RPE (4 ', day 20) cultures were treated with CM11 for 6 and 24 hours, and from these cultures and control samples Isolate RNA. Control samples were incubated in MEM5 for 6 hours.
結果: Results:
1. 對照組,0.4μg/μl,260/280=1.64,18μl 1. Control group, 0.4μg / μl, 260/280 = 1.64,18μl
2. 經CM11處理6h者,0.1μg/μl,260/280=3.28,18μl 2. If treated with CM11 for 6h, 0.1μg / μl, 260/280 = 3.28, 18μl
3. 經CM11處理24h者,0.21μg/μl,260/280=2.28,18μl 3. For 24h treated by CM11, 0.21μg / μl, 260/280 = 2.28, 18μl
自15-07-072 RPE之6’第21天培養物(經及未經CM11處理6或24小時)分離RNA。 RNA was isolated from the 6 'day 21 culture of 15-07-072 RPE (with and without CM11 treatment for 6 or 24 hours).
結果: Results:
未經CM11處理者,0.81μg/μl,260/280=1.21,18μl Without CM11 treatment, 0.81μg / μl, 260/280 = 1.21,18μl
經CM11處理6h者,0.52μg/μl,260/280=1.23,18μl For CM11 treatment for 6h, 0.52μg / μl, 260/280 = 1.23,18μl
經CM11處理24h者,1.07μg/μl,260/280=1.21,18μl For CM11 treatment for 24h, 1.07μg / μl, 260/280 = 1.21,18μl
自不同繼代之人類RPE培養物分離RNA,該等人類RPE培養物包括SD1「乾性」AMD L 5’第20天RPE培養物,其經或未經CM11處理6及24小時。其他人類RPE培養物包括正常15-08-074 4’第20天培養物、正常15-09-027 5’第7天培養物、及「濕性」AMD 15-10-021 4’第10天培養物。 RNA was isolated from different generations of human RPE cultures, including SD1 "dry" AMD L 5 'day 20 RPE cultures, with or without CM11 treatment for 6 and 24 hours. Other human RPE cultures include normal 15-08-074 4 'day 20 culture, normal 15-09-027 5' day 7 culture, and "wet" AMD 15-10-021 4 'day 10 Cultures.
結果: Results:
SD1 AMD L 5’第20天,0.1μg/μl,260/280=1.64,18μl SD1 AMD L 5 ’Day 20, 0.1μg / μl, 260/280 = 1.64,18μl
SD1 AMD L 5’第20天,經CM11處理6小時,0.07μg/μl,260/280=1.84,18μl SD1 AMD L 5 ’Day 20, treated with CM11 for 6 hours, 0.07μg / μl, 260/280 = 1.84,18μl
SD1 AMD L 5’第20天,經CM11處理24小時,0.09μg/μl,260/280=1.56,18μl SD1 AMD L 5 ’Day 20, treated with CM11 for 24 hours, 0.09μg / μl, 260/280 = 1.56,18μl
由於無法自SD1右(R)RPE分離RNA(因為生長不良且供應有限),已嘗試培育一些存活3’培養物。將存活3’培養物合併以分離出任何RNA(經及未經CM11處理24小時)。 Since it is not possible to isolate RNA from SD1 right (R) RPE (due to poor growth and limited supply), attempts have been made to breed some surviving 3 'cultures. The surviving 3 'cultures were combined to isolate any RNA (with and without CM11 treatment for 24 hours).
結果: Results:
1. SD1 AMD R 3’第56天RPE,0.4μg/μl,260/280=1.29,18μl 1. SD1 AMD R 3 ’Day 56 RPE, 0.4μg / μl, 260/280 = 1.29,18μl
2. SD1 AMD R 3’第56天RPE,經CM11處理24小時,0.25μg/μl,260/280=1.18 2. SD1 AMD R 3 ’Day 56 RPE, treated with CM11 for 24 hours, 0.25μg / μl, 260/280 = 1.18
自濕性AMD 15-10-021 RPE之4’第10天培養物(經及未經CM11處理6及24小時)分離RNA。 RNA was isolated from the 4 'day 10 culture of wet AMD 15-10-021 RPE (with and without CM11 treatment for 6 and 24 hours).
結果: Results:
未經CM11處理者,0.03μg/μl,260/280=0.58,18μl Without CM11 treatment, 0.03μg / μl, 260/280 = 0.58,18μl
經CM11處理6h者,0.09μg/μl,260/280=1.23,18μl After CM11 treatment for 6h, 0.09μg / μl, 260/280 = 1.23,18μl
經CM11處理24h者,0.11μg/μl,260/280=1.36,18μl After treated by CM11 for 24h, 0.11μg / μl, 260/280 = 1.36,18μl
自ND08333「AMD」RPE之2’第8天培養物(經及未經CM11處理6及24小時)分離RNA。 RNA was isolated from the 2 'day 8 culture of ND08333 "AMD" RPE (with and without CM11 treatment for 6 and 24 hours).
結果: Results:
1. 未經CM11處理者,0.04μg/μl,260/280=1.77,18μl 1. Without CM11 treatment, 0.04μg / μl, 260/280 = 1.77,18μl
2. 經CM11處理6h者,0.03μg/μl,260/280=1.77,18μl 2. For CM11 treatment for 6h, 0.03μg / μl, 260/280 = 1.77, 18μl
3. 經CM11處理24h者,0.04μg/μl,260/280=1.94,18μl 3. For CM11 treatment for 24h, 0.04μg / μl, 260/280 = 1.94,18μl
自ND08333 3’第106天和4’第97天RPE培養物(經及未經CM13處理6及24小時)分離RNA。 RNA was isolated from ND08333 3 'day 106 and 4' day 97 RPE cultures (with and without CM13 treatment for 6 and 24 hours).
結果: Results:
1. 3’無CM處理,0.02μg/μl,260/280=1.845,17μl 1. 3 ’without CM treatment, 0.02μg / μl, 260/280 = 1.845,17μl
2. 3’6h CM13,0.12μg/μl,260/280=2.022,17μl 2. 3’6h CM13, 0.12μg / μl, 260/280 = 2.022,17μl
3. 3’23h CM13,0.13μg/μl,260/280=2.393,17μl 3. 3’23h CM13, 0.13μg / μl, 260/280 = 2.393,17μl
4. 4’無CM處理,0.21μg/μl,260/280=2.027,17μl 4. 4 ’without CM treatment, 0.21μg / μl, 260/280 = 2.027, 17μl
5. 4’6h CM13,0.28μg/μl,260/280=2.056,17μl 5. 4’6h CM13, 0.28μg / μl, 260/280 = 2.056,17μl
6. 4’24h CM13,0.31μg/μl 260/280=2.064,17μl 6. 4’24h CM13, 0.31μg / μl 260/280 = 2.064,17μl
自ND08626 AMD RPE之1’第39天、2’第23天、及3’第5天培養物(經及未經CM5 10k處理6及24小時)分離RNA。 RNA was isolated from ND08626 AMD RPE 1 'day 39, 2' day 23, and 3 'day 5 cultures (with and without CM5 10k treatment for 6 and 24 hours).
結果: Results:
1. 1’第39天,未經CM5處理,0.02μg/μl,260/280=1.96,18μl 1. Day 39 of 1 ’, without CM5 treatment, 0.02μg / μl, 260/280 = 1.96,18μl
2. 1’第39天,經CM5處理6h,0.04μg/μl,260/280=3.03,18μl 2. Day 39 of 1 ’, treated with CM5 for 6h, 0.04μg / μl, 260/280 = 3.03, 18μl
3. 1’第39天,經CM5處理24h,0.03μg/μl,260/280=1.69,18μl 3. Day 39 on 1 ’, treated with CM5 for 24h, 0.03μg / μl, 260/280 = 1.69, 18μl
4. 2’第23天,未經CM5處理,0.1μg/μl,260/280=1.90,18μl 4. On day 23 of 2 ’, without CM5 treatment, 0.1μg / μl, 260/280 = 1.90,18μl
5. 2’第23天,經CM5處理6h,0.08μg/μl,260/280=1.92,18μl 5. On day 23 of 2 ’, treated with CM5 for 6h, 0.08μg / μl, 260/280 = 1.92,18μl
6. 2’第23天,經CM5處理24h,0.09μg/μl,260/280=1.84,18μl 6. On day 23 of 2 ’, treated with CM5 for 24h, 0.09μg / μl, 260/280 = 1.84,18μl
7. 3’第5天,未經CM5處理,0.08μg/μl,260/280=1.72,18μl 7. On the 5th day of 3 ’, without CM5 treatment, 0.08μg / μl, 260/280 = 1.72,18μl
8. 3’第5天,經CM5處理6h,0.16μg/μl,260/280=1.53,18μl 8. On the 5th day of 3 ’, treated with CM5 for 6h, 0.16μg / μl, 260/280 = 1.53,18μl
9. 3’第5天,經CM5處理24h,0.03μg/μl,260/280=1.87,18μl 9. On the 5th day of 3 ’, treated with CM5 for 24h, 0.03μg / μl, 260/280 = 1.87,18μl
自15-08-074 RPE之4’第17天培養物(經及未經CM11處理6或24小時)分離RNA。 RNA was isolated from the 4 'day 17 culture of 15-08-074 RPE (with and without CM11 treatment for 6 or 24 hours).
結果: Results:
1. 未經CM11處理者,0.15μg/μl,260/280=9.17,18μl 1. Without CM11 treatment, 0.15μg / μl, 260/280 = 9.17,18μl
2. 經CM11處理6h者,0.18μg/μl,260/280=1.32,18μl 2. For CM11 treatment for 6h, 0.18μg / μl, 260/280 = 1.32, 18μl
3. 經CM11處理24h者,0.18μg/μl,260/280=2.82,18μl 3. If treated with CM11 for 24h, 0.18μg / μl, 260/280 = 2.82, 18μl
自15-08-074 4’第20天RPE培養物(經及未經CM11處理6及24小時)分離RNA。 RNA was isolated from 15-08-074 4 'day 20 RPE cultures (with and without CM11 treatment for 6 and 24 hours).
結果: Results:
1. 未經CM11處理者,0.05μg/μl,260/280=0.86,18μl 1. Without CM11 treatment, 0.05μg / μl, 260/280 = 0.86,18μl
2. 經CM11處理6小時者,0.06μg/μl,260/280=1.60,18μl 2. For CM11 treatment for 6 hours, 0.06μg / μl, 260/280 = 1.60, 18μl
3. 經CM11處理24小時者,0.05μg/μl,260/280=1.30,18μl 3. After 24 hours of CM11 treatment, 0.05μg / μl, 260/280 = 1.30, 18μl
自許多正常及AMD RPE樣本(經及未經CM15處理6及24小時)製備RNA。藉由Nanodrop技術測定濃度。 RNA was prepared from many normal and AMD RPE samples (with and without CM15 treatment for 6 and 24 hours). The concentration was determined by Nanodrop technique.
結果: Results:
自許多正常及AMD RPE樣本(經及未經CM15處理6及24小時)製備RNA。藉由Nanodrop技術測定濃度。 RNA was prepared from many normal and AMD RPE samples (with and without CM15 treatment for 6 and 24 hours). The concentration was determined by Nanodrop technique.
結果: Results:
自NDRI AMD ND08626眼睛之1’第60天、2’第44天、及3’第26天RPE培養物(經及未經CM13處理6及24小時)分離RNA。 RNA was isolated from RPE cultures (with and without CM13 treatment for 6 and 24 hours) on day 1 'day 60, 2' day 44 and 3 'day 26 of NDRI AMD ND08626 eyes.
結果: Results:
自人類正常15-09-027 5’第7天培養物(經及未經CM11處理6及24小時)分離RNA。 RNA was isolated from human normal 15-09-027 5 'day 7 cultures (with and without CM11 treatment for 6 and 24 hours).
結果: Results:
自15-11-098 1’第50天及2’第14天RPE培養物(經及未經CM11處理24小時)分離RNA。 RNA was isolated from 15-11-098 1 'day 50 and 2' day 14 RPE cultures (with and without CM11 treatment for 24 hours).
結果: Results:
自15-11-098 2’第26天RPE(未經處理)及3’第12天RPE(經及未經CM11處理6及24小時)分離RNA。 RNA was isolated from 15-11-098 2 'day 26 RPE (untreated) and 3' day 12 RPE (with and without CM11 treatment for 6 and 24 hours).
結果: Results:
測試各種RTK配體對於RPE吞噬之作用。為進行測試,將重組RTK配體暴露於在實例6中獲得的各種RPE分離株。 The effect of various RTK ligands on RPE phagocytosis was tested. For testing, the recombinant RTK ligand was exposed to various RPE isolates obtained in Example 6.
重組人類BDNF(目錄號248-BD-025/CF,批號NG6515031)及人類GDNF(目錄號212-GD-010/CF,批號VQ2215081)係來自R&D Systems,Inc.,Minneapolis,MN。將其等以100μg/mL於無菌PBS中重構,經等分且冷凍於-70℃冰箱。重組人類HGF(目錄號GF116,批號2651986)係來自EMD Millipore Corp.,Temecula,CA。將其以0.5mg/mL於水中重構,經等分且冷凍於-20℃。 Recombinant human BDNF (catalog number 248-BD-025 / CF, batch number NG6515031) and human GDNF (catalog number 212-GD-010 / CF, batch number VQ2215081) are from R & D Systems, Inc., Minneapolis, MN. It was reconstituted in sterile PBS at 100 μg / mL, aliquoted and frozen in a refrigerator at -70 ° C. Recombinant human HGF (Cat. No. GF116, Lot No. 2651986) is from EMD Millipore Corp., Temecula, CA. It was reconstituted in water at 0.5 mg / mL, aliquoted and frozen at -20 ° C.
將AMD RPE細胞與各種濃度之重組人類BDNF(405pg/mL、2ng/mL、10ng/mL、50ng/mL、200ng/mL)、或重組人類HGF(792pg/mL、4.75ng/mL、28.5ng/mL、200ng/mL)、或重組 人類GDF(52.8pg/mL、422.4pg/mL、3.4ng/mL、27ng/mL、200ng/mL)培養24小時,且隨後在未改變培養基下進行吞噬檢定。經hUTC CM培養之AMD RPE細胞係用作為檢定之陽性對照。 Combine AMD RPE cells with various concentrations of recombinant human BDNF (405pg / mL, 2ng / mL, 10ng / mL, 50ng / mL, 200ng / mL), or recombinant human HGF (792pg / mL, 4.75ng / mL, 28.5ng / mL, 200 ng / mL), or recombinant human GDF (52.8 pg / mL, 422.4 pg / mL, 3.4 ng / mL, 27 ng / mL, 200 ng / mL) were cultured for 24 hours, and then phagocytosis assay was performed without changing the medium. The AMD RPE cell line cultured with hUTC CM was used as a positive control for the assay.
為了檢查RTK配體BDNF對於SD1「乾性」AMD RPE吞噬之作用,使SD1 AMD L RPE之3’第6天培養物在用或未用CM10或不同量BDNF培養之後,與2種正常人類RPE培養物進行吞噬檢定。測試結果顯示於表12-1。 In order to examine the effect of RTK ligand BDNF on SD1 "dry" AMD RPE phagocytosis, the 3 'day 6 culture of SD1 AMD L RPE was cultured with 2 normal human RPEs with or without CM10 or different amounts of BDNF Phagocytosis test. The test results are shown in Table 12-1.
已出現吞噬對於增加水準之BDNF的劑量反應,除了2ng/ml結果。SD1 AMD RPE相較於正常(15-09-027及15-08-074)RPE低的吞噬水準再次經過確認。CM10之添加增加了SD1 AMD RPE之吞噬水準。 There has been a dose response of phagocytosis to increased levels of BDNF, except for the 2ng / ml result. The low phagocytosis level of SD1 AMD RPE compared to normal (15-09-027 and 15-08-074) RPE was confirmed again. The addition of CM10 increases the phagocytosis level of SD1 AMD RPE.
為了檢查RTK配體HGF對於SD1「乾性」AMD RPE吞噬之作用,使SD1 AMD L RPE之4’第10天培養物在用或未用CM10或不同量HGF培養之後,與2種正常人類RPE培養物進行吞噬檢定。測試結果顯示於表12-2。 To examine the effect of RTK ligand HGF on the phagocytosis of SD1 "dry" AMD RPE, the 4 'day 10 culture of SD1 AMD L RPE was cultured with 2 normal human RPEs with or without CM10 or different amounts of HGF Phagocytosis test. The test results are shown in Table 12-2.
HGF並未顯示對於SD1 AMD 4’RPE吞噬之劑量效應,不過其大致上增加吞噬。SD1 AMD RPE之吞噬水準再次低於正常(15-09-027及15-08-074)RPE,而CM10之添加增加了吞噬水準。 HGF does not show a dose effect on SD1 AMD 4'RPE phagocytosis, but it generally increases phagocytosis. The phagocytosis level of SD1 AMD RPE was again lower than normal (15-09-027 and 15-08-074) RPE, while the addition of CM10 increased the phagocytosis level.
表12-2. RTK配體HGF對於SD1「乾性」AMD L 4’RPE吞噬之作
為了檢查RTK配體GDNF對於AMD RPE(San Diego #1 L)吞噬之作用,除了經及未經CM10以外,執行SD1 L 3’第11天RPE吞噬對於增加水準之GDNF(52.8pg/ml、422.4pg/ml、3.4ng/ml、27ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使 15-09-027 N 3’第11天及15-08-074 N 3’第21天之RPE進行對各為2×106個的15-10-021上帶ROS之8h吞噬分析。獲得在大部分此等劑量反應實驗中所出現之典型模式的結果。亦即:1)AMD RPE(SD1 L)吞噬對於RTK配體(GDNF)之明確劑量反應;2)AMD RPE(SD1 L)相較於正常RPE(15-09-027、15-08-074)降低的吞噬水準;及3)AMD RPE(SD1 L)之降低的吞噬水準藉由CM(CM10)處理之正常化。 In order to examine the effect of RTK ligand GDNF on AMD RPE (San Diego # 1 L) phagocytosis, in addition to and without CM10, perform SD1 L 3 'Day 11 RPE phagocytosis for increased levels of GDNF (52.8pg / ml, 422.4 pg / ml, 3.4ng / ml, 27ng / ml, 200ng / ml) dose response. As for the normal control group, 15-09-027 N 3 'day 11 and 15-08-074 N 3' day 21 RPE were also carried on 15-10-021 with 2 × 10 6 each 8h phagocytosis analysis. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (SD1 L) engulfed a clear dose response to RTK ligand (GDNF); 2) AMD RPE (SD1 L) compared to normal RPE (15-09-027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (SD1 L) is normalized by CM (CM10) treatment.
為了檢查RTK配體HGF對於AMD RPE(15-10-021,「濕性」)吞噬之作用,除了經及未經CM10以外,執行15-10-021 3’第13天RPE吞噬對於增加水準之HGF(792pg/ml、4.75ng/ml、28.5ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使15-09-027 N 3’第26天及15-08-074 N 3’第36天之RPE進行對各為2×106個的15-10-021上帶ROS之8h吞噬分析。獲得略微非典型模式的結果。亦即:1)AMD RPE(15-10-021)吞噬水準對於RTK配體HGF之明確增加,但無劑量效應;2)AMD RPE(15-10-021)相較於正常RPE(15-09-027、15-08-074)降低的吞噬水準;及3)AMD RPE(15-10-021)之降低的吞噬水準藉由CM(CM10)處理之正常化(參見圖31)。 In order to examine the effect of RTK ligand HGF on AMD RPE (15-10-021, "wet") phagocytosis, in addition to and without CM10, perform 15-10-021 3 'day 13 RPE phagocytosis for increased levels The dose response of HGF (792pg / ml, 4.75ng / ml, 28.5ng / ml, 200ng / ml). As for the normal control group, 15-09-027 N 3 'day 26 and 15-08-074 N 3' day 36 RPE were also performed on 15-10-021 of 2 × 10 6 each with ROS 8h phagocytosis analysis. Get slightly atypical results. That is: 1) AMD RPE (15-10-021) phagocytosis level clearly increased RTK ligand HGF, but no dose effect; 2) AMD RPE (15-10-021) compared to normal RPE (15-09 -027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) is normalized by CM (CM10) treatment (see FIG. 31).
為了檢查RTK配體BDNF對於AMD RPE(15-10-021,「濕性」)吞噬之作用,除了經及未經CM13以外,執行15-10-021 4’第206天RPE吞噬對於增加水準之BDNF(405pg/ml、2ng/ml、10ng/ml、50ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使15-09-027 N5’第203天及15-08-074 N 4’第216天之RPE進行對各為5×105個的15-11-098上帶ROS之8h吞噬分析。獲得略微典型模式的結果。亦即:1)AMD RPE(15-10-021)吞噬水準對於RTK配體BDNF之些許劑量效應;2)AMD RPE(15-10-021)相較於正常RPE(15-09-027;15-08-074並未作用)降低的吞噬水準;及3)AMD RPE(15-10-021)之降低的吞噬水準藉由CM(CM13)處理之正常化(參見圖32)。 In order to check the effect of RTK ligand BDNF on AMD RPE (15-10-021, "wet") phagocytosis, in addition to being with or without CM13, perform 15-10-021 4 'day 206 RPE phagocytosis for increasing levels Dose response of BDNF (405pg / ml, 2ng / ml, 10ng / ml, 50ng / ml, 200ng / ml). As for the normal control group, RPE on 15-09-027 N5 'day 203 and 15-08-074 N 4' day 216 were also tested on 15-11-098 with 5 × 10 5 pieces each 8h phagocytosis analysis. Get slightly typical results. That is: 1) AMD RPE (15-10-021) phagocytosis level has some dose effect on RTK ligand BDNF; 2) AMD RPE (15-10-021) compared to normal RPE (15-09-027; 15 -08-074 did not work) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) was normalized by CM (CM13) treatment (see Figure 32).
為了檢查RTK配體GDNF對於AMD RPE(15-10-021,「濕性」)吞噬之作用,除了經及未經CM11以外,執行15-10-021 4’第7天RPE吞噬對於增加水準之GDNF(52.8pg/ml、422.4pg/ml、3.4ng/ml、27ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使15-09-027 N 4’第33天及15-08-074 N 4’第17天之RPE進行對各為2×106個的15-10-021上帶ROS之8h吞噬分析。獲得在大部分此等劑量反應實驗中所出現之典型模式的結果。亦 即:1)AMD RPE(15-10-021)吞噬對於RTK配體(GDNF)之明確劑量反應;2)AMD RPE(15-10-021)相較於正常RPE(15-09-027、15-08-074)降低的吞噬水準;及3)AMD RPE(15-10-021)之降低的吞噬水準藉由CM(CM11)處理之正常化(參見圖33)。 In order to examine the effect of RTK ligand GDNF on AMD RPE (15-10-021, "wet") phagocytosis, in addition to and without CM11, perform 15-10-021 4 'day 7 RPE phagocytosis for increased levels Dose response of GDNF (52.8pg / ml, 422.4pg / ml, 3.4ng / ml, 27ng / ml, 200ng / ml). As for the normal control group, 15-09-027 N 4 'day 33 and 15-08-074 N 4' day 17 RPE were also carried on the 15-10-021 with 2 × 10 6 each. 8h phagocytosis analysis. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (15-10-021) engulfed a clear dose response to RTK ligand (GDNF); 2) AMD RPE (15-10-021) compared to normal RPE (15-09-027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) is normalized by CM (CM11) treatment (see Figure 33).
為了檢查RTK配體BDNF對於AMD RPE(ND08333)吞噬之作用,除了經及未經CM11以外,執行ND08333 2’第20天RPE吞噬對於增加水準之BDNF(405pg/ml、2ng/ml、10ng/ml、50ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使15-11-098 N 3’第12天及15-08-074 N 4’第74天之RPE進行對各為1×106個的15-11-098上帶ROS之8h吞噬分析。獲得略微典型模式的結果。亦即:1)AMD RPE(ND08333)吞噬水準對於RTK配體BDNF之些許劑量效應;2)AMD RPE(ND08333)相較於正常RPE(15-11-098及15-08-074)降低的吞噬水準;及3)AMD RPE(ND08333)之降低的吞噬水準藉由CM(CM11)處理之正常化(參見圖34)。 In order to examine the effect of RTK ligand BDNF on AMD RPE (ND08333) phagocytosis, in addition to and without CM11, perform ND08333 2 'day 20 RPE phagocytosis on increased levels of BDNF (405pg / ml, 2ng / ml, 10ng / ml , 50ng / ml, 200ng / ml) dose response. As for the normal control group, 15-11-098 N 3 'day 12 and 15-08-074 N 4' day 74 RPE were also performed on 15-11-098 with 1 × 10 6 ROS on each 8h phagocytosis analysis. Get slightly typical results. That is: 1) AMD RPE (ND08333) phagocytosis level has some dose effect on RTK ligand BDNF; 2) AMD RPE (ND08333) phagocytosis is reduced compared to normal RPE (15-11-098 and 15-08-074) Level; and 3) The reduced phagocytosis level of AMD RPE (ND08333) is normalized by CM (CM11) treatment (see Figure 34).
為了檢查RTK配體HGF對於AMD RPE(ND08333)吞噬之作用,除了經及未經CM11以外,執行ND08333 2’第22天RPE吞噬對於增加水準之HGF(792pg/ml、4.75ng/ml、28.5ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使15-11-098 N 3’第 14天及15-08-074 N 4’第76天之RPE進行對各為1×106個的15-11-098上帶ROS之8h吞噬分析。獲得非典型模式的結果。亦即:1)AMD RPE(ND08333)吞噬水準對於RTK配體HGF之些許劑量反應;2)AMD RPE(ND08333)相較於正常RPE(15-11-098、15-08-074)降低的吞噬水準;但3)沒有AMD RPE(ND08333)之降低的吞噬水準藉由CM(CM11)處理之正常化,其可能歸因於缺乏CM11活性(參見圖35)。 In order to examine the effect of RTK ligand HGF on AMD RPE (ND08333) phagocytosis, in addition to and without CM11, perform ND08333 2 'day 22 RPE phagocytosis on increased levels of HGF (792pg / ml, 4.75ng / ml, 28.5ng / ml, 200ng / ml) dose response. As for the normal control group, 15-11-098 N 3 'day 14 and 15-08-074 N 4' day 76 RPE were also performed on 15-11-098 of 1 × 10 6 each with ROS 8h phagocytosis analysis. Obtain results for atypical patterns. That is: 1) AMD RPE (ND08333) phagocytic level responds to some dose of RTK ligand HGF; 2) AMD RPE (ND08333) reduced phagocytosis compared to normal RPE (15-11-098, 15-08-074) Level; but 3) the reduced phagocytosis level without AMD RPE (ND08333) is normalized by CM (CM11) treatment, which may be attributed to lack of CM11 activity (see Figure 35).
為了檢查RTK配體GDNF對於AMD RPE(ND08333)吞噬之作用,除了經及未經CM11以外,執行ND08333 2’第15天RPE吞噬對於增加水準之GDNF(52.8pg/ml、422.4pg/ml、3.4ng/ml、27ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使15-11-098 N 2’第21天及15-08-074 N 4’第69天之RPE進行對各為1×106個的15-11-098上帶ROS之8h吞噬分析。獲得在大部分此等劑量反應實驗中所出現之典型模式的結果。亦即:1)AMD RPE(ND08333)吞噬對於RTK配體(GDNF)之明確劑量反應;2)AMD RPE(ND08333)相較於正常RPE(15-11-098、15-08-074)降低的吞噬水準;及3)AMD RPE(ND08333)之降低的吞噬水準藉由CM(CM11)處理之正常化(參見圖36)。 In order to examine the effect of RTK ligand GDNF on AMD RPE (ND08333) phagocytosis, in addition to and without CM11, perform ND08333 2 'Day 15 RPE phagocytosis for increased levels of GDNF (52.8pg / ml, 422.4pg / ml, 3.4 ng / ml, 27ng / ml, 200ng / ml) dose response. As for the normal control group, 15-11-098 N 2 'on the 21st day and 15-08-074 N 4' on the 69th day of RPE were performed on 15-11-098 with 1 × 10 6 ROS on each 8h phagocytosis analysis. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (ND08333) phagocytosis of RTK ligand (GDNF) a clear dose response; 2) AMD RPE (ND08333) compared to normal RPE (15-11-098, 15-08-074) reduced Phagocytosis level; and 3) The reduced phagocytosis level of AMD RPE (ND08333) is normalized by CM (CM11) treatment (see Figure 36).
為了檢查RTK配體GDNF對於AMD RPE(ND08626)吞噬之作用,除了經及未經CM5 10k以外,執行ND08626 2’第28天RPE吞噬對於增加水準之GDNF(52.8pg/ml、422.4pg/ml、3.4ng/ml、27ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使15-09-027 N 5’第116天及15-11-098 N 3’第67天之RPE進行對各為0.5×106個的15-11-098上帶ROS之8h吞噬分析。獲得在大部分此等劑量反應實驗中所出現之典型模式的結果。亦即:1)AMD RPE(ND08626)吞噬對於RTK配體(GDNF)之明確劑量反應;2)AMD RPE(ND08626)相較於正常RPE(15-09-027、15-11-098)降低的吞噬水準;及3)AMD RPE(ND08626)之降低的吞噬水準藉由CM(CM5 10k)處理之正常化(參見圖37)。 In order to examine the effect of RTK ligand GDNF on AMD RPE (ND08626) phagocytosis, in addition to and without CM5 10k, perform ND08626 2 'day 28 RPE phagocytosis for increased levels of GDNF (52.8pg / ml, 422.4pg / ml, 3.4ng / ml, 27ng / ml, 200ng / ml) dose response. As for the normal control group, 15-11-098 N 5 'day 116 and 15-11-098 N 3' day 67 RPE were also performed on 15-11-098 with 0.5 × 10 6 each carrying ROS 8h phagocytosis analysis. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) The clear dose response of AMD RPE (ND08626) phagocytosis to RTK ligand (GDNF); 2) The decrease in AMD RPE (ND08626) compared to normal RPE (15-09-027, 15-11-098) Phagocytosis level; and 3) The reduced phagocytosis level of AMD RPE (ND08626) is normalized by CM (CM5 10k) treatment (see Figure 37).
為了檢查RTK配體BDNF對於AMD RPE(ND08626)吞噬之作用,除了經及未經CM5 10k以外,執行ND08626 2’第25天RPE吞噬對於增加水準之BDNF(405pg/ml、2ng/ml、10ng/ml、50ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使15-09-027 N 5’第113天及15-11-098 N 3’第64天之RPE進行對各為0.5×106個的15-11-098上帶ROS之8h吞噬分析。獲得略微典型模式的結果。亦即:1)AMD RPE(Nd08626)吞噬水準對於RTK配體BDNF之中等劑量效應;2)AMD RPE(ND08626)相較於正常RPE(15-09-027、15-11-098)降低的吞噬水準;及3)AMD RPE (ND08626)之降低的吞噬水準藉由CM(CM5 10k)處理之正常化(參見圖38)。 In order to examine the effect of RTK ligand BDNF on AMD RPE (ND08626) phagocytosis, in addition to and without CM5 10k, perform ND08626 2 'day 25 RPE phagocytosis on increased levels of BDNF (405pg / ml, 2ng / ml, 10ng / ml, 50ng / ml, 200ng / ml) dose response. As for the normal control group, 15-09-027 N 5 'day 113 and 15-11-098 N 3' day 64 RPE were also performed on each 15-11-098 with 0.5 × 10 6 ROS 8h phagocytosis analysis. Get slightly typical results. That is: 1) AMD RPE (Nd08626) phagocytosis level is equal to the effect of RTK ligand BDNF; 2) AMD RPE (ND08626) reduced phagocytosis compared to normal RPE (15-09-027, 15-11-098) Level; and 3) Normalization of the reduced phagocytosis level of AMD RPE (ND08626) by CM (CM5 10k) treatment (see Figure 38).
為了檢查RTK配體HGF對於AMD RPE(ND08626)吞噬之作用,除了經及未經CM5 10k以外,執行ND08626 3’第11天RPE吞噬對於增加水準之HGF(792pg/ml、4.75ng/ml、28.5ng/ml、200ng/ml)的劑量反應。至於正常對照組,亦使15-09-027 N 5’第117天及15-11-098 N 3’第68天之RPE進行對各為0.5×106個的15-11-098上帶ROS之8h吞噬分析。獲得略微非典型模式的結果。亦即:1)AMD RPE(ND08626)吞噬水準對於RTK配體HGF之輕微劑量效應跡象;2)AMD RPE(ND08626)相較於正常RPE(15-09-027、15-11-098)降低的吞噬水準;及3)AMD RPE(ND08626)之降低的吞噬水準藉由CM(CM5 10k)處理之正常化(參見圖39)。 In order to examine the effect of RTK ligand HGF on phagocytosis of AMD RPE (ND08626), in addition to and without CM5 10k, perform ND08626 3 'day 11 RPE phagocytosis on increased levels of HGF (792pg / ml, 4.75ng / ml, 28.5 ng / ml, 200ng / ml) dose response. As for the normal control group, 15-09-027 N 5 'day 117 and 15-11-098 N 3' day 68 RPE were also performed on 15-11-098 with 0.5 × 10 6 ROS on each 8h phagocytosis analysis. Get slightly atypical results. That is: 1) AMD RPE (ND08626) phagocytic level has a slight dose effect on RTK ligand HGF; 2) AMD RPE (ND08626) is lower than normal RPE (15-09-027, 15-11-098) Phagocytosis level; and 3) The reduced phagocytosis level of AMD RPE (ND08626) is normalized by CM (CM5 10k) treatment (see Figure 39).
亦測試各種橋分子對於RPE吞噬之作用。為進行測試,將重組橋分子暴露於在實例6中獲得的各種RPE分離株。 The effect of various bridge molecules on RPE phagocytosis was also tested. For testing, the recombinant bridge molecule was exposed to various RPE isolates obtained in Example 6.
重組人類MFG-E8(目錄號2767-MF-050,批號MPP2415081)、重組人類TSP-1(目錄號3074-TH-050,批號MVF4114111)、重組人類TSP-2(目錄號1635-T2-050,批號HUZ2014121)全部皆獲自R&D Systems,Inc.,Minneapolis,MN。個別蛋白質儲備液之重構係根據供應商之資料表:重組人類MFG-E8、TSP-1及TSP-2分別以100μg/mL於無菌PBS中重構。重組人類Gas6以100μg/mL重構於無菌水中。經重構之儲備液經等分且冷凍於-70℃冰箱。 Recombinant human MFG-E8 (catalog number 2767-MF-050, batch number MPP2415081), recombinant human TSP-1 (catalog number 3074-TH-050, batch number MVF4114111), recombinant human TSP-2 (catalog number 1635-T2-050, Lot number HUZ2014121) were all obtained from R & D Systems, Inc., Minneapolis, MN. The reconstitution of individual protein stock solutions was based on the supplier's data sheet: recombinant human MFG-E8, TSP-1 and TSP-2 were reconstituted in sterile PBS at 100 μg / mL, respectively. Recombinant human Gas6 was reconstituted in sterile water at 100 μg / mL. The reconstituted stock solution was aliquoted and frozen in the refrigerator at -70 ° C.
將ROS以hUTC CM對照培養基(具有10% FBS及1% P/S之DMEM:F12培養基)或hUTC CM在37℃下於CO2細胞培養器中預培養24h。同時,ROS係在37℃下於CO2細胞培養器中,於對照培養基中預培養24h,該對照培養基含有各種濃度之人類重組MFG-E8(15.5ng/mL、31ng/mL、62ng/mL、124ng/mL)、TSP-1(152ng/mL、304ng/mL、608ng/mL、1216ng/mL)、或TSP-2(8.8ng/mL、26.4ng/mL、79.2ng/mL、237.6ng/mL)。在培養之後,將ROS離心沉降,再懸浮於MEM5(具有5% FBS之MEM培養基)中,且在MEM5存在之情況下饋至失養性RPE細胞以用於吞噬檢定。至於對照組,將單獨正常RPE或將單獨失養性RPE培養於MEM20(具有20% FBS之MEM培養基)中,隨後在未處理之ROS (再懸浮於MEM20中且饋至RPE細胞)存在之情況下更換成MEM5以用於吞噬檢定。 ROS were pre-incubated with hUTC CM control medium (DMEM with 10% FBS and 1% P / S: F12 medium) or hUTC CM in a CO 2 cell culture incubator at 37 ° C for 24 h. At the same time, the ROS line was pre-cultured in a CO 2 cell culture incubator at 37 ° C for 24h in a control medium containing various concentrations of human recombinant MFG-E8 (15.5ng / mL, 31ng / mL, 62ng / mL, 124ng / mL), TSP-1 (152ng / mL, 304ng / mL, 608ng / mL, 1216ng / mL), or TSP-2 (8.8ng / mL, 26.4ng / mL, 79.2ng / mL, 237.6ng / mL) ). After culturing, the ROS was centrifuged to settle, resuspended in MEM5 (MEM medium with 5% FBS), and fed to dystrophic RPE cells in the presence of MEM5 for phagocytosis assay. As for the control group, normal RPE alone or deprived RPE alone were cultured in MEM20 (MEM medium with 20% FBS), and then in the presence of untreated ROS (resuspended in MEM20 and fed to RPE cells) Replace with MEM5 for phagocytosis test.
為了檢查橋分子(MFG-E8)對於AMD RPE(SD1 L)吞噬之作用,將各為5×106個的15-09-027上帶ROS以增加水準之橋分子MFG-E8(15.5ng/ml、31ng/ml、62ng/ml、124ng/ml)處理(亦經及未經CM10處理)24小時,而且經處理及未經處理之ROS係用於AMD SD1 L 4’第10天RPE及正常對照組15-09-027 N 4’第8天和15-08-074 N 4’第24天RPE之8h吞噬檢定。獲得在大部分此等劑量反應實驗中所出現之典型模式的結果。亦即:1)AMD RPE(SD1 L)吞噬對於經橋分子(MFG-E8)處理之ROS的明確劑量反應;2)AMD RPE(SD1 L)相較於正常RPE(15-09-027、15-08-074)降低的吞噬水準;及3)AMD RPE(SD1 L)之降低的吞噬水準藉由以CM(CM10)處理ROS之正常化(參見圖40)。 In order to examine the effect of the bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (SD1 L), 15-09-027 of 5 × 10 6 each were provided with ROS to increase the level of the bridge molecule MFG-E8 (15.5ng / ml, 31ng / ml, 62ng / ml, 124ng / ml) treatment (also treated with and without CM10) for 24 hours, and the treated and untreated ROS are used for AMD SD1 L 4 'RPE on day 10 and normal The control group 15-09-027 N 4 'day 8 and 15-08-074 N 4' day 24 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (SD1 L) engulfed a clear dose response to ROS treated with bridge molecules (MFG-E8); 2) AMD RPE (SD1 L) compared to normal RPE (15-09-027, 15 -08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (SD1 L) is normalized by treatment of ROS with CM (CM10) (see FIG. 40).
為了檢查橋分子(Tsp 1)對於AMD RPE(SD1 L)吞噬之作用,將各為2×106個的15-09-027上帶ROS以增加水準之橋分子Tsp 1(152ng/ml、304ng/ml、608ng/ml、1216ng/ml)處理(亦經及未經CM10處理)24小時,而且經處理及未經處理之ROS係用於AMD SD1 L 4’第16天RPE及正常對照組15-09-027 N 4’第14天 和15-08-074 N 4’第29天RPE之8h吞噬檢定。獲得略微非典型模式的結果。亦即:1)AMD RPE(SD1 L)吞噬水準對於經橋分子(Tsp 1)處理之ROS的明確增加,但無劑量效應;2)AMD RPE(SD1 L)相較於正常RPE(15-09-027、15-08-074)降低的吞噬水準;及3)AMD RPE(SD1 L)之降低的吞噬水準藉由以CM(CM10)處理ROS之正常化(參見圖41)。 In order to check the effect of the bridge molecule (Tsp 1) on the phagocytosis of AMD RPE (SD1 L), 2 × 10 6 pieces of 15-09-027 were added with ROS to increase the level of bridge molecule Tsp 1 (152ng / ml, 304ng / ml, 608ng / ml, 1216ng / ml) treatment (also with and without CM10 treatment) for 24 hours, and the treated and untreated ROS were used for AMD SD1 L 4 'RPE on day 16 and normal control group 15 -09-027 N 4 'day 14 and 15-08-074 N 4' day 29 RPE 8h phagocytosis test. Get slightly atypical results. That is: 1) AMD RPE (SD1 L) phagocytosis level has a clear increase in ROS treated with bridge molecules (Tsp 1), but there is no dose effect; 2) AMD RPE (SD1 L) compared to normal RPE (15-09 -027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (SD1 L) is normalized by treatment of ROS with CM (CM10) (see FIG. 41).
為了檢查橋分子(Tsp 2)對於AMD RPE(SD1 L)吞噬之作用,將各為2×106個的15-09-027上帶ROS以增加水準之橋分子Tsp 2(8.8ng/ml、26.4ng/ml、79.2ng/ml、237.6ng/ml)處理(亦經及未經CM10處理)24小時,而且經處理及未經處理之ROS係用於AMD SD1 L 4’第14天RPE及正常對照組15-09-027 N 4’第13天和15-08-074 N 4’第28天RPE之8h吞噬檢定。獲得在大部分此等劑量反應實驗中所出現之典型模式的結果。亦即:1)AMD RPE(SD1 L)吞噬對於經橋分子(Tsp 2)處理之ROS的明確劑量反應;2)AMD RPE(SD1 L)相較於正常RPE(15-09-027、15-08-074)降低的吞噬水準;及3)AMD RPE(SD1 L)之降低的吞噬水準藉由以CM(CM10)處理ROS之正常化(參見圖42)。 In order to check the effect of the bridge molecule (Tsp 2) on the phagocytosis of AMD RPE (SD1 L), 15-09-027 with 2 × 10 6 each were added with ROS to increase the level of the bridge molecule Tsp 2 (8.8ng / ml, 26.4ng / ml, 79.2ng / ml, 237.6ng / ml) treatment (also with and without CM10 treatment) for 24 hours, and the treated and untreated ROS were used for AMD SD1 L 4 '14th day RPE and The normal control group 15-09-027 N 4 'day 13 and 15-08-074 N 4' day 28 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (SD1 L) engulfed a clear dose response to ROS treated with bridge molecules (Tsp 2); 2) AMD RPE (SD1 L) compared to normal RPE (15-09-027, 15- 08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (SD1 L) is normalized by treatment of ROS with CM (CM10) (see Figure 42).
為了檢查橋分子(Tsp 1)對於AMD RPE(15-10-021,「濕性」)吞噬之作用,將各為5×106個的15-09-027上帶ROS以增加水準之橋分子Tsp 1(152ng/ml、304ng/ml、608ng/ml、1216ng/ml)處理(亦經及未經CM10處理)24小時,而且經處理及未經處理之ROS係用於AMD 15-10-021 3’第15天RPE及正常對照組15-09-027 N 3’第28天和15-08-074 N 3’第38天RPE之8h吞噬檢定。獲得在大部分此等劑量反應實驗中所出現之相當典型模式的結果。亦即:1)AMD RPE(15-10-021)吞噬對於經橋分子(Tsp 1)處理之ROS的些許劑量反應;2)AMD RPE(15-10-021)相較於正常RPE(15-09-027、15-08-074)降低的吞噬水準;及3)AMD RPE(15-10-021)之降低的吞噬水準藉由以CM(CM10)處理ROS之正常化(參見圖43)。 In order to check the effect of the bridge molecule (Tsp 1) on the phagocytosis of AMD RPE (15-10-021, "wetness"), 15-09-027 with 5 × 10 6 each were added with ROS to increase the level of bridge molecules Tsp 1 (152ng / ml, 304ng / ml, 608ng / ml, 1216ng / ml) treatment (also treated with and without CM10) for 24 hours, and treated and untreated ROS are used for AMD 15-10-021 RPE of 3'day 15 and normal control group 15-09-027 N 3 'day 28 and 15-08-074 N 3' day 38 RPE 8h phagocytosis test. Obtain results in a fairly typical model that has appeared in most of these dose response experiments. That is: 1) AMD RPE (15-10-021) engulfed in a dose response to ROS treated with bridge molecule (Tsp 1); 2) AMD RPE (15-10-021) compared to normal RPE (15- 09-027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) is normalized by treatment of ROS with CM (CM10) (see FIG. 43).
為了檢查橋分子(MFG-E8)對於AMD RPE(15-10-021「濕性」)吞噬之作用,將各為2×106個的15-09-027上帶ROS以增加水準之橋分子MFG-E8(15.5ng/ml、31ng/ml、62ng/ml、124ng/ml)處理(亦經及未經CM10處理)24小時,而且經處理及未經處理之ROS係用於AMD 15-10-021 3’第12天RPE及正常對照組15-09-027 N 3’第25天和15-08-074 N 3’第35天RPE之8h吞噬檢定。獲得在大部分此等劑量反應實驗中所出現之接近典型模式的結 果。亦即:1)AMD RPE(15-10-021)吞噬對於經橋分子(MFG-E8)處理之ROS的劑量反應(雖然為低水準);2)AMD RPE(15-10-021)相較於正常RPE(15-09-027、15-08-074)降低的吞噬水準;及3)AMD RPE(15-10-021)之降低的吞噬水準藉由以CM(CM10)處理ROS之正常化(參見圖44)。 In order to check the effect of the bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (15-10-021 "wetness"), 2 × 10 6 pieces of 15-09-027 were added with ROS to increase the level of bridge molecules MFG-E8 (15.5ng / ml, 31ng / ml, 62ng / ml, 124ng / ml) treatment (also with and without CM10 treatment) for 24 hours, and the treated and untreated ROS are used for AMD 15-10 -021 3 'Day 12 RPE and normal control group 15-09-027 N 3' Day 25 and 15-08-074 N 3 'Day 35 RPE 8h phagocytosis test. Obtain results close to typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (15-10-021) phagocytosis dose-response (although low level) to ROS treated with bridge molecule (MFG-E8); 2) AMD RPE (15-10-021) compared Reduced phagocytosis level in normal RPE (15-09-027, 15-08-074); and 3) Reduced phagocytosis level in AMD RPE (15-10-021) normalization by treating ROS with CM (CM10) (See Figure 44).
為了檢查橋分子(Tsp 2)對於AMD RPE(15-10-021「濕性」)吞噬之作用,將各為2×106個的15-09-027上帶ROS以增加水準之橋分子Tsp 2(8.8ng/ml、26.4ng/ml、79.2ng/ml、237.6ng/ml)處理(亦經及未經CM10處理)24小時,而且經處理及未經處理之ROS係用於AMD 15-10-021 3’第13天RPE及正常對照組15-09-027 N 3’第26天和15-08-074 N 3’第36天RPE之8h吞噬檢定。獲得在大部分此等劑量反應實驗中所出現之典型模式的結果。亦即:1)AMD RPE(15-10-021)吞噬對於經橋分子(Tsp 2)處理之ROS的明確劑量反應;2)AMD RPE(15-10-021)相較於正常RPE(15-09-027、15-08-074)降低的吞噬水準;及3)AMD RPE(15-10-021)之降低的吞噬水準藉由以CM(CM10)處理ROS之正常化(參見圖45)。 In order to check the effect of the bridge molecule (Tsp 2) on the phagocytosis of AMD RPE (15-10-021 "wetness"), 2 × 10 6 pieces of 15-09-027 were added with ROS to increase the level of bridge molecule Tsp 2 (8.8ng / ml, 26.4ng / ml, 79.2ng / ml, 237.6ng / ml) treatment (also with and without CM10 treatment) for 24 hours, and the treated and untreated ROS are used for AMD 15- 10-021 3 'day 13 RPE and normal control group 15-09-027 N 3' day 26 and 15-08-074 N 3 'day 36 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (15-10-021) engulfed a clear dose response to ROS treated with bridge molecules (Tsp 2); 2) AMD RPE (15-10-021) compared to normal RPE (15-021) 09-027, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (15-10-021) is normalized by treatment of ROS with CM (CM10) (see FIG. 45).
為了檢查橋分子(MFG-E8)對於AMD RPE(ND08333)吞噬之作用,將各為1×106個的15-11-098上帶ROS以增加水準之 橋分子MFG-E8(15.5ng/ml、31ng/ml、62ng/ml、124ng/ml)處理(亦經及未經CM11處理)24小時,而且經處理及未經處理之ROS係用於AMD ND08333 2’第20天RPE及正常對照組15-11-098 N 3’第12天和15-08-074 N 4’第74天RPE之8h吞噬檢定。獲得在大部分此等劑量反應實驗中所出現之接近典型模式的結果。亦即:1)AMD RPE(ND08333)吞噬對於經橋分子(MFG-E8)處理之ROS的中等劑量反應;2)AMD RPE(ND08333)相較於正常RPE(15-11-098、15-08-074)降低的吞噬水準;及3)AMD RPE(ND08333)之降低的吞噬水準藉由以CM(CM11)處理ROS之正常化(參見圖46)。 In order to check the effect of the bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (ND08333), 15-11-098 of 1 × 10 6 each were provided with ROS to increase the level of bridge molecule MFG-E8 (15.5ng / ml , 31ng / ml, 62ng / ml, 124ng / ml) treatment (also treated with and without CM11) for 24 hours, and the treated and untreated ROS were used for AMD ND08333 2 'day 20 RPE and normal control group 15-11-098 N 3 'day 12 and 15-08-074 N 4' day 74 RPE 8h phagocytosis test. Obtain results close to typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (ND08333) phagocytosis response to ROS treated with bridge molecule (MFG-E8); 2) AMD RPE (ND08333) compared to normal RPE (15-11-098, 15-08 -074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08333) is normalized by treatment of ROS with CM (CM11) (see Figure 46).
為了檢查橋分子(Tsp 1)對於AMD RPE(ND08333)吞噬之作用,將各為0.5×106個的15-11-098上帶ROS以增加水準之橋分子Tsp 1(152ng/ml、304ng/ml、608ng/ml、1216ng/ml)處理(亦經及未經CM13處理)24小時,而且經處理及未經處理之ROS係用於AMD ND08333 3’第56天RPE及正常對照組15-11-098 N 3’第61天和15-08-074 N 4’第124天RPE之8h吞噬檢定。獲得略微非典型模式的結果。亦即:1)AMD RPE(ND08333)吞噬水準對於經橋分子(Tsp 1)處理之ROS的輕微增加,但無真正劑量效應;2)AMD RPE(ND08333)相較於正常RPE(15-11-098、15-08-074)降低的吞噬水準;及3)AMD RPE(ND08333)之降低的吞噬水準藉由以CM(CM13)處理ROS之正常化(參見圖47)。 In order to check the effect of the bridge molecule (Tsp 1) on the phagocytosis of AMD RPE (ND08333), 15-11-098 with 0.5 × 10 6 each were added with ROS to increase the level of the bridge molecule Tsp 1 (152ng / ml, 304ng / ml, 608ng / ml, 1216ng / ml) treatment (also treated with and without CM13) for 24 hours, and the treated and untreated ROS were used for AMD ND08333 3 'day 56 RPE and normal control group 15-11 -098 N 3 'Day 61 and 15-08-074 N 4' Day 124 RPE 8h phagocytosis test. Get slightly atypical results. That is: 1) AMD RPE (ND08333) phagocytosis level slightly increased ROS treated with bridge molecule (Tsp 1), but no real dose effect; 2) AMD RPE (ND08333) compared to normal RPE (15-11- 098, 15-08-074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08333) is normalized by treatment of ROS with CM (CM13) (see FIG. 47).
為了檢查橋分子(Tsp 2)對於AMD RPE(ND08333)吞噬之作用,將各為1×106個的15-11-098上帶ROS以增加水準之橋分子Tsp 2(8.8ng/ml、26.4ng/ml、79.2ng/ml、237.6ng/ml)處理(亦經及未經CM11處理)24小時,而且經處理及未經處理之ROS係用於AMD ND08333 2’第20天RPE及正常對照組15-11-098 N 3’第12天和15-08-074 N 4’第74天RPE之8h吞噬檢定。獲得在大部分此等劑量反應實驗中所出現之典型模式的結果。亦即:1)AMD RPE(ND08333)吞噬對於經橋分子(Tsp 2)處理之ROS的明確劑量反應;2)AMD RPE(ND08333)相較於正常RPE(15-11-098、15-08-074)降低的吞噬水準;及3)AMD RPE(ND08333)之降低的吞噬水準藉由以CM(CM11)處理ROS之正常化(參見圖48)。 In order to check the effect of the bridge molecule (Tsp 2) on the phagocytosis of AMD RPE (ND08333), 15-11-098 with 1 × 10 6 each were added with ROS to increase the level of the bridge molecule Tsp 2 (8.8ng / ml, 26.4 ng / ml, 79.2ng / ml, 237.6ng / ml) treatment (also treated with and without CM11) for 24 hours, and the treated and untreated ROS were used for AMD ND08333 2 'day 20 RPE and normal controls Group 15-11-098 N 3 'day 12 and 15-08-074 N 4' day 74 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (ND08333) engulfed a clear dose response to ROS treated with bridge molecules (Tsp 2); 2) AMD RPE (ND08333) compared to normal RPE (15-11-098, 15-08- 074) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08333) is normalized by treatment of ROS with CM (CM11) (see Figure 48).
為了檢查橋分子(MFG-E8)對於AMD RPE(ND08626)吞噬之作用,將各為0.5×106個的15-11-098上帶ROS以增加水準之橋分子MFG-E8(15.5ng/ml、31ng/ml、62ng/ml、124ng/ml)處理(亦經及未經CM5 10k處理)24小時,而且經處理及未經處理之ROS係用於AMD ND08626 3’第5天RPE及正常對照組15-09-027 N 5’第111天和15-11-098 N 3’第62天RPE之8h吞噬檢定。獲得在大部分此等劑量反應實驗中所出現之典型模式的結果。亦即:1) AMD RPE(ND08626)吞噬對於經橋分子(MFG-E8)處理之ROS的明確劑量反應;2)AMD RPE(ND08626)相較於正常RPE(15-09-027、15-11-098)降低的吞噬水準;及3)AMD RPE(ND08626)之降低的吞噬水準藉由以CM(CM5 10k)處理ROS之正常化(參見圖49)。 In order to examine the effect of the bridge molecule (MFG-E8) on the phagocytosis of AMD RPE (ND08626), 15-11-098 with 0.5 × 10 6 each were added with ROS to increase the level of bridge molecule MFG-E8 (15.5ng / ml , 31ng / ml, 62ng / ml, 124ng / ml) treatment (also with and without CM5 10k treatment) for 24 hours, and the treated and untreated ROS were used for AMD ND08626 3 'day 5 RPE and normal controls Group 15-09-027 N 5 'day 111 and 15-11-098 N 3' day 62 RPE 8h phagocytosis test. Obtain the results of the typical patterns that appear in most of these dose response experiments. That is: 1) AMD RPE (ND08626) engulfed a clear dose response to ROS treated with bridge molecule (MFG-E8); 2) AMD RPE (ND08626) compared to normal RPE (15-09-027, 15-11 -098) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08626) is normalized by treatment of ROS with CM (CM5 10k) (see Figure 49).
為了檢查橋分子(Tsp 1)對於AMD RPE(ND08626)吞噬之作用,將各為0.5×106個的15-11-098上帶ROS以增加水準之橋分子Tsp 1(152ng/ml、304ng/ml、608ng/ml、1216ng/ml)處理(亦經及未經CM5 10k處理)24小時,而且經處理及未經處理之ROS係用於AMD ND08626 3’第14天RPE及正常對照組15-09-027 N 5’第120天和15-11-098 N 3’第71天RPE之8h吞噬檢定。獲得在大部分此等劑量反應實驗中所出現之相當典型模式的結果。亦即:1)AMD RPE(ND08626)吞噬對於經橋分子(Tsp 1)處理之ROS的中等劑量反應;2)AMD RPE(ND08626)相較於正常RPE(15-09-027、15-11-098)降低的吞噬水準;及3)AMD RPE(ND08626)之降低的吞噬水準藉由以CM(CM5 10k)處理ROS之正常化(參見圖50)。 In order to check the effect of the bridge molecule (Tsp 1) on the phagocytosis of AMD RPE (ND08626), 15-11-098 with 0.5 × 10 6 each were added with ROS to increase the level of the bridge molecule Tsp 1 (152ng / ml, 304ng / ml, 608ng / ml, 1216ng / ml) treatment (also with and without CM5 10k treatment) for 24 hours, and the treated and untreated ROS were used for AMD ND08626 3 '14th day RPE and normal control group 15- 9-027 N 5 'day 120 and 15-11-098 N 3' day 71 RPE 8h phagocytosis test. Obtain results in a fairly typical model that has appeared in most of these dose response experiments. That is: 1) AMD RPE (ND08626) phagocytosis response to ROS treated with bridging molecule (Tsp 1); 2) AMD RPE (ND08626) compared to normal RPE (15-09-027, 15-11- 098) Reduced phagocytosis level; and 3) Reduced phagocytosis level of AMD RPE (ND08626) is normalized by treatment of ROS with CM (CM5 10k) (see FIG. 50).
為了檢查橋分子(Tsp 2)對於AMD RPE(ND08626)吞噬之作用,將各為0.5×106個的15-11-098上帶ROS以增加水準之 橋分子Tsp 2(8.8ng/ml、26.4ng/ml、79.2ng/ml、237.6ng/ml)處理(亦經及未經CM5 10k處理)24小時,而且經處理及未經處理之ROS係用於AMD ND08626 3’第5天RPE及正常對照組15-09-027 N 5’第111天和15-11-098 N 3’第62天RPE之8h吞噬檢定。獲得略微非典型模式的結果。亦即:1)AMD RPE(ND08626)吞噬對於經橋分子(Tsp 2)處理之ROS的明確劑量反應;2)AMD RPE(ND08626)相較於正常RPE(15-09-027、15-11-098)降低的吞噬水準;及3)AMD RPE(ND08626)之降低的吞噬水準藉由以CM(CM5 10k)處理ROS之部分正常化,其可能歸因於CM活性衰退(參見圖51)。 In order to check the effect of the bridge molecule (Tsp 2) on the phagocytosis of AMD RPE (ND08626), 15-11-098 with 0.5 × 10 6 each were added with ROS to increase the level of the bridge molecule Tsp 2 (8.8ng / ml, 26.4 ng / ml, 79.2ng / ml, 237.6ng / ml) treatment (also with and without CM5 10k treatment) for 24 hours, and the treated and untreated ROS are used for AMD ND08626 3 'day 5 RPE and normal The control group 15-09-027 N 5 'day 111 and 15-11-098 N 3' day 62 RPE 8h phagocytosis test. Get slightly atypical results. That is: 1) AMD RPE (ND08626) engulfed a clear dose response to ROS treated with bridge molecule (Tsp 2); 2) AMD RPE (ND08626) compared to normal RPE (15-09-027, 15-11- 098) Reduced phagocytosis level; and 3) The reduced phagocytosis level of AMD RPE (ND08626) is normalized by the partial treatment of ROS with CM (CM5 10k), which may be attributed to the decline in CM activity (see Figure 51).
實例6至13證明下列各點: Examples 6 to 13 demonstrate the following points:
A. 確認AMD RPE相較於正常RPE的吞噬功能下降(乾性與濕性AMD兩者皆是如此)。 A. Confirm that AMD RPE has reduced phagocytic function compared to normal RPE (both dry and wet AMD).
B. 確認hUTC CM對於人類RPE吞噬功能的刺激作用,該人類RPE包括AMD及甚至正常RPE(對於前者之作用較後者為多),使得AMD RPE中減少的吞噬功能在大部分情況下經正常化。 B. Confirm the stimulating effect of hUTC CM on the phagocytosis of human RPE, which includes AMD and even normal RPE (the former has more effects than the latter), so that the reduced phagocytosis in AMD RPE is normalized in most cases .
C. 確認如於大鼠RCS RPE中所出現,RTK配體及橋分子對於AMD RPE吞噬功能呈劑量依賴性方式的刺激作用。 C. Confirm that the RTK ligand and the bridge molecule stimulate the AMD RPE phagocytosis in a dose-dependent manner as seen in rat RCS RPE.
儘管所涉及之疾病及物種性質有別(基於RCS大鼠模型中MERTK基因缺陷的視網膜變性、以及老年人的主要人類眼睛疾病AMD),在實例6至13中存在其於RPE吞噬功能缺陷及hUTC CM救援吞噬缺陷之有效性的類似處。hUTC CM校正吞噬功能異常的有效性,證明hUTC作為用於視網膜變性及AMD之治療的效力。 Despite the differences in the diseases and species involved (retinal degeneration based on the MERTK gene defect in the RCS rat model, and AMD, the main human eye disease in the elderly), there are defects in RPE phagocytosis and hUTC in Examples 6 to 13 CM rescues similarities in the effectiveness of phagocytosis defects. The effectiveness of hUTC CM to correct abnormal phagocytosis proves the effectiveness of hUTC as a treatment for retinal degeneration and AMD.
此實例描述自胎盤及臍帶組織製備產後衍生細胞。在足月或不足月生產後獲得產後臍帶及胎盤。細胞係收集自五位不同捐贈者之臍帶及胎盤組織。測試不同的細胞分離方法產生具有以下潛能之細胞的能力:1)分化成具有不同表型之細胞的潛能,此為幹細胞共有之特徵;或2)提供可用於其他細胞及組織之營養因子之潛能。 This example describes the preparation of postpartum-derived cells from placenta and umbilical cord tissue. Obtain the postpartum umbilical cord and placenta after full-term or insufficient-term delivery. The cell line was collected from the umbilical cord and placental tissue of five different donors. Test the ability of different cell separation methods to produce cells with the following potentials: 1) the potential to differentiate into cells with different phenotypes, which is a common feature of stem cells; or 2) the potential to provide nutritional factors that can be used in other cells and tissues .
臍細胞分離:臍帶係獲自國家疾病研究交換中心(NDRI,Philadelphia,Pa.)。組織係在正常分娩後獲得。細胞分離規程係在層流櫃(laminar flow hood)中無菌執行。為了移除血液及碎屑,將臍帶在磷酸鹽緩衝液(PBS;Invitrogen,Carlsbad,Calif.)中於抗黴劑及抗生素(100單位/毫升的青黴素、100微克/毫升的鏈黴素、0.25微克/毫升的兩性黴素B)存在下來清洗。接著在150cm2組織培養盤上並且於50毫升的培養基(DMEM-低葡萄糖或DMEM-高葡萄糖;Invitrogen)存在下機械解離組織,直到將組織切碎為細泥。將切碎的組織轉移至50毫升錐形管(每管約5公克的組織)。 Umbilical cell isolation: The umbilical cord was obtained from the National Disease Research Exchange Center (NDRI, Philadelphia, Pa.). The tissue system is obtained after normal delivery. The cell separation procedure is performed aseptically in a laminar flow hood. To remove blood and debris, place the umbilical cord in phosphate buffer (PBS; Invitrogen, Carlsbad, Calif.) With antimycotics and antibiotics (100 units / ml penicillin, 100 μg / ml streptomycin, 0.25 Amphotericin B) in the presence of micrograms / ml was washed down. The tissue was then mechanically dissociated on a 150 cm 2 tissue culture dish and in the presence of 50 ml of medium (DMEM-low glucose or DMEM-high glucose; Invitrogen) until the tissue was minced into fine mud. Transfer the minced tissue to a 50 ml conical tube (approximately 5 grams of tissue per tube).
隨後將組織於DMEM-低葡萄糖培養基或DMEM-高葡萄糖培養基中消化,各培養基含有如上文所述之抗黴劑及抗生素。在一些實驗中,使用膠原蛋白酶與分散酶的酶混合物(「C:D」)(膠原蛋白酶(Sigma,St Louis,Mo.),500個單位/毫升;與分散酶(Invitrogen),50個單位/毫升,在DMEM-低葡萄糖培養基中)。在其他實驗中,使用膠原蛋白酶、分散酶與玻尿酸酶的混合物(「C:D:H」)(膠原蛋白酶,500個單位/毫升;分散酶,50個單位/毫升;與玻尿酸酶(Sigma),5個單位/毫升,在DMEM-低葡萄糖中)。使含有組織、培養基與消化酶的錐形管在37℃下在迴轉式振盪器(Environ,Brooklyn,N.Y)中以225rpm培養2小時。 The tissue is then digested in DMEM-low glucose medium or DMEM-high glucose medium, each medium containing the antifungal agent and antibiotic as described above. In some experiments, an enzyme mixture of collagenase and dispase ("C: D") (collagenase (Sigma, St Louis, Mo.), 500 units / ml; and dispase (Invitrogen), 50 units were used / Ml in DMEM-low glucose medium). In other experiments, a mixture of collagenase, dispase and hyaluronidase ("C: D: H") (collagenase, 500 units / ml; dispase, 50 units / ml; and hyaluronidase (Sigma) , 5 units / ml in DMEM-low glucose). A conical tube containing tissue, culture medium, and digestive enzymes was incubated at 37 ° C. for 2 hours at 225 rpm in a rotary shaker (Environ, Brooklyn, N.Y).
在消化後,將組織以150xg離心5分鐘,且將上清液吸出。將團塊再懸浮於20毫升的生長培養基(DMEM-低葡萄糖(Invitrogen)、15百分比(v/v)胎牛血清(FBS;特級牛血清(defined bovine serum);批號AND18475;Hyclone,Logan,Utah)、0.001%(v/v)2-巰基乙醇(Sigma)、每100毫升1毫升如上文所述之抗生素/抗黴劑。使細胞懸浮液通過70微米尼龍細胞濾器(BD Biosciences)過濾。使額外5毫升包含生長培養基的潤洗液通過濾器。接著使細胞懸浮液通過40微米的尼龍細胞濾器(BD Biosciences),然後用額外5毫升的生長培養基潤洗液來趕出。 After digestion, the tissue was centrifuged at 150 x g for 5 minutes, and the supernatant was aspirated. Resuspend the pellet in 20 ml of growth medium (DMEM-low glucose (Invitrogen), 15% (v / v) fetal bovine serum (FBS; special bovine serum); batch number AND18475; Hyclone, Logan, Utah ), 0.001% (v / v) 2-mercaptoethanol (Sigma), 1 ml per 100 ml of antibiotic / antifungal agent as described above. The cell suspension was filtered through a 70 micron nylon cell filter (BD Biosciences). An additional 5 ml of rinse medium containing growth medium was passed through the filter. The cell suspension was then passed through a 40 micron nylon cell strainer (BD Biosciences) and then extra 5 ml of growth medium rinse was used to drive out.
將濾液再懸浮於生長培養基(總體積50毫升)中然後以150xg離心5分鐘。將上清液吸出然後將細胞再懸浮於50毫升的新鮮生長培養基中。將此過程再重複兩次。 The filtrate was resuspended in growth medium (total volume 50 ml) and then centrifuged at 150 x g for 5 minutes. Aspirate the supernatant and resuspend the cells in 50 ml of fresh growth medium. Repeat this process two more times.
在最終離心後,將上清液吸出然後將細胞團塊再懸浮於5毫升的新鮮生長培養基中。使用台盼藍染色來測定存活細胞數目。接著在標準條件下培養細胞。 After the final centrifugation, the supernatant was aspirated and the cell pellet was resuspended in 5 ml of fresh growth medium. Trypan blue staining was used to determine the number of viable cells. The cells are then cultured under standard conditions.
將分離自臍帶的細胞以5,000個細胞/cm2接種於經明膠塗布T-75cm2培養瓶(Corning Inc.,Corning,N.Y.)上並且於具有如上文所述之抗生素/抗黴劑的生長培養基中。2天後(在各種實驗中,細胞培養2至4天),將用過的培養基自瓶吸出。用PBS清洗細胞三次以去除碎屑與血衍生細胞。接著用生長培養基補充細胞並讓其生長至長滿(自繼代0至繼代1約10日)。在後續繼代時(自繼代1至2等等),細胞在4至5日後達到次長滿(75至85百分比長滿)。針對這些後續繼代,以5000個細胞/cm2接種細胞。使細胞在加濕培養器中於5百分比二氧化碳及大氣氧氣、37℃下生長。 Cells isolated from the umbilical cord were seeded at 5,000 cells / cm 2 on gelatin-coated T-75cm 2 culture flasks (Corning Inc., Corning, NY) and on growth medium with antibiotics / antifungals as described above in. After 2 days (in various experiments, the cells were cultured for 2 to 4 days), the used medium was aspirated from the flask. Wash cells three times with PBS to remove debris and blood-derived cells. The cells were then supplemented with growth medium and allowed to grow to fullness (from passage 0 to passage 1 for about 10 days). At subsequent passages (since passages 1 to 2, etc.), the cells reach the second overgrowth after 4 to 5 days (75 to 85 percent overgrowth). For these subsequent passages, cells were seeded at 5000 cells / cm 2 . The cells were grown in a humidified incubator at 37% carbon dioxide and atmospheric oxygen.
胎盤細胞分離:胎盤組織係獲自NDRI(Philadelphia,Pa.)。該些組織係來自孕婦且在正常外科分娩時獲得。胎盤細胞係如臍細胞分離所述分離。 Isolation of placental cells: Placental tissue lines were obtained from NDRI (Philadelphia, Pa.). These tissues are from pregnant women and are obtained during normal surgical delivery. The placental cell line is isolated as described for umbilical cell isolation.
以下實例應用於自胎盤組織分離母體衍生及新生兒衍生之分開細胞群。 The following examples apply to the separation of maternally-derived and neonate-derived separate cell populations from placental tissue.
細胞分離規程係在層流櫃(laminar flow hood)中無菌執行。將胎盤組織在磷酸鹽緩衝液(PBS;Invitrogen,Carlsbad,Calif.)中於抗黴劑及抗生素(如上文所述)之存在下清洗,以移除血液及碎屑。隨後將胎盤組織解剖成三區:頂線(新生兒側或態樣)、中線(混合的細胞分離新生兒及母體)及底線(母體側或態樣)。 The cell separation procedure is performed aseptically in a laminar flow hood. Placental tissue was washed in phosphate buffer (PBS; Invitrogen, Carlsbad, Calif.) In the presence of antifungal agents and antibiotics (as described above) to remove blood and debris. Subsequently, the placental tissue was dissected into three areas: top line (neonatal side or appearance), midline (mixed cells separating neonate and mother) and bottom line (maternal side or appearance).
將分開的區個別於具有抗生素/抗黴劑之PBS中清洗數次以進一步移除血及碎屑。接著在150cm2組織培養盤上於50毫升的DMEM-低葡萄糖存在下機械解離各區,直到將各區切碎為細泥。將泥轉移至50毫升錐形管中。各管含有大致5公克組織。將組織於含有抗黴劑及抗生素(100U/毫升青黴素、100微克/毫升鏈黴素、0.25微克/毫升兩性黴素B)及消化酶之DMEM-低葡萄糖或DMEM-高葡萄糖培養基中消化。在一些實驗中,使用膠原蛋白酶與分散酶的酶混合物(「C:D」),其於DMEM-低葡萄糖培養基中含有500個單位/毫升之膠原蛋白酶(Sigma,St Louis,Mo.)與50個單位/毫升之分散酶(Invitrogen)。在其他實驗中,使用膠原蛋白酶、分散酶與玻尿酸酶的混合物(C:D:H)(膠原蛋白酶,500個單位/毫升;分散酶,50個單位/毫升;與玻尿酸酶(Sigma),5個單位/毫升,在DMEM-低葡萄糖中)。使含有組織、培養基與消化酶的錐形管在37℃下在迴轉式振盪器(Environ,Brooklyn,NY)中以225rpm培養2小時。 The separated areas were washed several times in PBS with antibiotics / antimycotics to further remove blood and debris. Next, the zones were mechanically dissociated on a 150 cm 2 tissue culture plate in the presence of 50 ml of DMEM-low glucose until the zones were chopped into fine mud. Transfer the mud to a 50 ml conical tube. Each tube contains approximately 5 grams of tissue. The tissues were digested in DMEM-low glucose or DMEM-high glucose medium containing antifungal agents and antibiotics (100 U / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin B) and digestive enzymes. In some experiments, an enzyme mixture of collagenase and dispase ("C: D") was used, which contained 500 units / ml collagenase (Sigma, St Louis, Mo.) and 50 in DMEM-low glucose medium Units / ml of dispase (Invitrogen). In other experiments, a mixture of collagenase, dispase and hyaluronidase (C: D: H) (collagenase, 500 units / ml; dispase, 50 units / ml; and hyaluronidase (Sigma), 5 Units / ml in DMEM-low glucose). A conical tube containing tissue, culture medium, and digestive enzymes was incubated at 225 rpm for 2 hours at 37 ° C in a rotary shaker (Environ, Brooklyn, NY).
在消化後,將組織以150xg離心5分鐘,將所得上清液吸出。將團塊再懸浮於20毫升的具有青黴素/鏈黴素/兩性黴素B之生長培養基中。使細胞懸浮液過濾通過70微米的尼龍細胞濾器(BD Biosciences),用額外5毫升的生長培養基潤洗液趕出。使總細胞懸浮液通過40微米的尼龍細胞濾器(BD Biosciences),接著用額外5毫升的生長培養基作為潤洗液。 After digestion, the tissue was centrifuged at 150 x g for 5 minutes, and the resulting supernatant was aspirated. The pellet was resuspended in 20 ml of growth medium with penicillin / streptomycin / amphoterin B. The cell suspension was filtered through a 70 micron nylon cell strainer (BD Biosciences) and chased out with an additional 5 ml of growth medium rinse. The total cell suspension was passed through a 40 micron nylon cell strainer (BD Biosciences), followed by an additional 5 ml of growth medium as a rinse.
將濾液再懸浮於生長培養基(總體積50毫升)中然後以150xg離心5分鐘。將上清液吸出然後將細胞團塊再懸浮於50 毫升的新鮮生長培養基中。將此過程再重複兩次。在最終離心後,將上清液吸出然後將細胞團塊再懸浮於5毫升的新鮮生長培養基中。使用台盼藍排除測試來測定細胞計數。接著在標準條件下培養細胞。 The filtrate was resuspended in growth medium (total volume 50 ml) and then centrifuged at 150 x g for 5 minutes. Aspirate the supernatant and resuspend the cell pellet in 50 ml of fresh growth medium. Repeat this process two more times. After the final centrifugation, the supernatant was aspirated and the cell pellet was resuspended in 5 ml of fresh growth medium. The trypan blue exclusion test was used to determine the cell count. The cells are then cultured under standard conditions.
LIBERASE細胞分離:於具有LIBERASE(Boehringer Mannheim Corp.,Indianapolis,Ind.)(每毫升2.5毫克,Blendzyme 3;Roche Applied Sciences,Indianapolis,Ind.)及玻尿酸酶(5個單位/毫升,Sigma)之DMEM-低葡萄糖培養基中將細胞自臍組織分離。組織消化與細胞分離係如以上之其他蛋白酶消化所述,但使用LIBERASE/玻尿酸酶混合物代替C:D或C:D:H酶混合物。用LIBERASE消化組織會使來自產後組織迅速擴增的細胞族群得以分離出來。 LIBERASE cell isolation: in DMEM with LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) (2.5 mg per ml, Blendzyme 3; Roche Applied Sciences, Indianapolis, Ind.) And hyaluronidase (5 units / ml, Sigma) -Separation of cells from umbilical tissue in low glucose medium. Tissue digestion and cell separation are as described above for other protease digestions, but use LIBERASE / hyaluronidase mixture instead of C: D or C: D: H enzyme mixture. Digestion of tissues with LIBERASE allows the separation of rapidly expanding cell populations from postpartum tissues.
使用其他酶組合的細胞分離:對於使用不同酶組合以自臍帶分離出細胞的程序進行比較。用於消化比較之酶包括:i)膠原蛋白酶;ii)分散酶;iii)玻尿酸酶;iv)膠原蛋白酶:分散酶混合物(C:D);v)膠原蛋白酶:玻尿酸酶混合物(C:H);vi)分散酶:玻尿酸酶混合物(D:H);與vii)膠原蛋白酶:分散酶:玻尿酸酶混合物(C:D:H)。觀察利用這些不同酶消化條件之細胞分離的差異(表14-1)。 Cell separation using other enzyme combinations: Comparison of procedures using different enzyme combinations to isolate cells from the umbilical cord. Enzymes used for digestion comparison include: i) collagenase; ii) dispase; iii) hyaluronidase; iv) collagenase: dispase mixture (C: D); v) collagenase: hyaluronidase mixture (C: H) ; Vi) dispase: hyaluronidase mixture (D: H); and vii) collagenase: dispase: hyaluronidase mixture (C: D: H). Observe the differences in cell separation using these different enzyme digestion conditions (Table 14-1).
自臍帶中之殘餘血液分離細胞:進行其他嘗試以藉由不同方式將細胞池自臍帶分離。在一個例子中,將臍帶切片然後用生長培養基清洗以排出血塊與凝膠狀材料。收集血、凝膠狀材料與生長培養基之混合物然後以150xg離心。將團塊再懸浮並接種於經 明膠塗布培養瓶上之生長培養基中。透過這些實驗,分離出會迅速擴增的細胞群。 Separation of cells from residual blood in the umbilical cord: Other attempts were made to separate the cell pool from the umbilical cord by different means. In one example, the umbilical cord is sliced and then washed with growth medium to expel blood clots and gel-like material. The mixture of blood, gel-like material and growth medium was collected and centrifuged at 150 x g. The pellet was resuspended and inoculated into growth medium on gelatin-coated culture flasks. Through these experiments, a population of cells that would rapidly expand was isolated.
自臍帶血分離細胞:亦自得自NDR1之臍帶血樣本分離細胞。此處所使用之分離規程為Ho等人之國際專利申請案WO 2003/025149中者(Ho,T.W.,等人,「Cell Populations Which Co-Express CD49C and CD90」,申請號PCT/US02/29971)。將臍帶血(NDRI,Philadelphia PA)樣本(分別為50毫升與10.5毫升)與裂解緩衝液(經過濾器滅菌之155mM氯化銨、10毫莫耳碳酸氫鉀、0.1毫莫耳EDTA、緩衝至pH 7.2(所有組分皆來自Sigma,St.Louis,Mo.))混合。使細胞以1:20臍帶血對裂解緩衝液的比例溶解。使所生成的細胞懸浮液渦動5秒鐘,然後在環境溫度下培養2分鐘。將溶解產物離心(以200xg離心10分鐘)。將細胞團塊再懸浮於完全最低必需培養基(Gibco,Carlsbad,Calif.)中,該培養基含有10百分比胎牛血清(Hyclone,Logan Utah)、4毫莫耳麩醯胺酸(Mediatech,Herndon,Va.)、每100毫升100個單位青黴素與每100毫升100微克鏈黴素(Gibco,Carlsbad,Calif.)。將再懸浮之細胞離心(以200xg離心10分鐘)、將上清液吸出然後在完全培養基中清洗細胞團塊。將細胞直接接種至T75培養瓶(Corning,N.Y.)、T75經層黏蛋白塗布培養瓶或T175經纖連蛋白素塗布培養瓶(均來自Becton Dickinson,Bedford,Mass.)。 Isolate cells from cord blood: Isolate cells from a cord blood sample obtained from NDR1. The separation protocol used here is Ho International's International Patent Application WO 2003/025149 (Ho, TW, et al., "Cell Populations Which Co-Express CD49C and CD90", application number PCT / US02 / 29971). Cord blood (NDRI, Philadelphia PA) samples (50 ml and 10.5 ml, respectively) and lysis buffer (155 mM ammonium chloride sterilized by filter, 10 mM potassium bicarbonate, 0.1 mM EDTA, buffered to pH 7.2 (all components are from Sigma, St. Louis, Mo.)) mixed. The cells were lysed at a ratio of 1:20 cord blood to lysis buffer. The resulting cell suspension was vortexed for 5 seconds and then incubated at ambient temperature for 2 minutes. The lysate was centrifuged (centrifuged at 200 x g for 10 minutes). The cell pellet was resuspended in complete minimum essential medium (Gibco, Carlsbad, Calif.) Containing 10% fetal bovine serum (Hyclone, Logan Utah), 4 millimolar branamide (Mediatech, Herndon, Va .), 100 units of penicillin per 100 ml and 100 micrograms of streptomycin per 100 ml (Gibco, Carlsbad, Calif.). Centrifuge the resuspended cells (centrifugation at 200 x g for 10 minutes), aspirate the supernatant and wash the cell pellet in complete medium. Cells were directly seeded into T75 flasks (Corning, NY), T75 laminin coated flasks or T175 fibronectin coated flasks (both from Becton Dickinson, Bedford, Mass.).
使用不同酶組合及生長條件分離細胞:為了要判定細胞群是否可在不同條件下分離並在分離後是否可立即在各種條件下 擴增,根據以上提供之程序使用C:D:H的酶組合,將細胞在含有或未含0.001百分比(v/v)2-巰基乙醇(Sigma,St.Louis,Mo.)的生長培養基中消化。在多種條件下接種如此分離之胎盤衍生細胞。所有細胞皆於青黴素/鏈黴素存在下生長。(表14-2)。 Separation of cells using different enzyme combinations and growth conditions: To determine whether the cell population can be isolated under different conditions and whether it can be expanded immediately under various conditions after separation, use the C: D: H enzyme combination according to the procedure provided above The cells were digested in growth medium with or without 0.001% (v / v) 2-mercaptoethanol (Sigma, St. Louis, Mo.). The placenta-derived cells thus isolated were seeded under various conditions. All cells are grown in the presence of penicillin / streptomycin. (Table 14-2).
使用不同酶組合及生長條件分離細胞:在所有條件下,細胞在繼代0與1之間附著及擴增良好(表14-2)。條件5至8與13至16中之細胞皆顯示在接種後良好增生多達4次繼代,將這些細胞在此時點凍存且存庫。 Cells were separated using different enzyme combinations and growth conditions: under all conditions, the cells attached and expanded well between passages 0 and 1 (Table 14-2). The cells in conditions 5 to 8 and 13 to 16 all showed good proliferation up to 4 passages after inoculation, and these cells were frozen and stored at this point.
使用不同酶組合之細胞分離:C:D:H之組合提供最佳分離後細胞產率,並且比起其他條件在培養中產生擴增更多代的細胞(表14-1)。單獨使用膠原蛋白酶或玻尿酸酶無法獲得可擴增的細胞群。未試圖判定此結果是否專屬於所測試之膠原蛋白。 Cell separation using a combination of different enzymes: the combination of C: D: H provides the best post-separation cell yield and produces cells that are expanded in more generations in culture than other conditions (Table 14-1). Collagen protease or hyaluronidase alone cannot obtain an expandable cell population. No attempt was made to determine whether this result was specific to the collagen tested.
使用不同酶組合及生長條件分離細胞:在所有針對酶消化及生長所測試的條件下,細胞皆在繼代0與1之間附著及擴增良好(表14-2)。實驗條件5至8與13至16中之細胞皆在接種後良好增生多達4次繼代,將這些細胞在此時點凍存。所有細胞皆經凍存以備進一步研究。 Cells were isolated using different enzyme combinations and growth conditions: under all conditions tested for enzyme digestion and growth, cells adhered and expanded well between passages 0 and 1 (Table 14-2). The cells in experimental conditions 5 to 8 and 13 to 16 all proliferated well up to 4 passages after seeding, and these cells were frozen at this point. All cells were cryopreserved for further study.
自臍帶中之殘餘血分離細胞:已成核細胞附著且生長快速。以流動式細胞測量術分析這些細胞,而這些細胞與透過酶消化所得之細胞相似。 Isolate cells from residual blood in the umbilical cord: nucleated cells attach and grow rapidly. These cells were analyzed by flow cytometry, and these cells were similar to those obtained by enzyme digestion.
自臍帶血分離細胞:製劑含有紅血球及血小板。在前3個星期期間沒有已成核細胞之附著與分裂。在接種後3個星期更換培養基但仍未觀察到細胞附著與生長。 Separation of cells from cord blood: The preparation contains red blood cells and platelets. There was no attachment and division of nucleated cells during the first 3 weeks. The culture medium was changed 3 weeks after inoculation but cell attachment and growth were still not observed.
總結:可使用膠原蛋白酶(基質金屬蛋白酶)、分散酶(中性蛋白酶)、及玻尿酸酶(分解玻尿酸之黏液分解酶)的酶組合,可有效地將細胞群自臍帶及胎盤組織衍生出來。亦可使用名為Blendzyme之LIBERASE。特定言之,亦可使用Blendzyme 3並搭配玻尿酸酶來分離細胞,Blendzyme 3為膠原蛋白酶(4 Wunsch單位/g)與嗜熱菌蛋白酶(1714酪蛋白單位/g)。當在明膠塗布之塑膠上的生長培養基中培養時,這些細胞迅速擴增許多個繼代。 Summary: Enzyme combinations of collagenase (matrix metalloproteinase), dispase (neutral protease), and hyaluronidase (mucolytic enzyme that breaks down hyaluronic acid) can be used to effectively derive cell populations from the umbilical cord and placental tissue. LIBERASE called Blendzyme can also be used. In particular, Blendzyme 3 can also be used in conjunction with hyaluronidase to isolate cells. Blendzyme 3 is collagenase (4 Wunsch units / g) and thermolysin (1714 casein units / g). When cultured in growth medium on gelatin-coated plastics, these cells rapidly expanded many passages.
亦自臍帶中的殘餘血分離出細胞,但該殘餘血並非臍帶血。自組織洗出的血塊中之所以存在於所用之條件下會貼附與生長之細胞,可能是因為細胞在解剖過程期間釋出。 Cells were also isolated from the residual blood in the umbilical cord, but the residual blood was not cord blood. The reason why the blood clot washed out of the tissue exists and adheres and grows under the conditions used may be because the cells are released during the dissection process.
細胞療法所用之細胞系較佳為均質性並且不含任何汙染細胞型。細胞療法所用之細胞應具有正常染色體數目(46)及結 構。為了要識別出均質性且不含非產後組織來源之細胞的胎盤及臍帶衍生細胞系,對於細胞樣本之核型進行分析。 The cell line used for cell therapy is preferably homogeneous and does not contain any contaminating cell types. Cells used in cell therapy should have a normal chromosome number (46) and structure. In order to identify homogeneous placenta and umbilical cord-derived cell lines that do not contain cells of non-postpartum tissue origin, the karyotype of the cell samples was analyzed.
在含有青黴素/鏈黴素之生長培養基中培養來自男新生兒之產後組織的PPDC。選用來自男新生兒(X,Y)的產後組織以能夠區別新生兒衍生細胞與母體衍生細胞(X,X)。將細胞以每平方公分5,000細胞接種於T25培養瓶(Corning Inc.,Corning,N.Y.)中的培養基中並且擴增至80%長滿。將含有細胞之T25培養瓶用生長培養基填充至頸部。由快遞員將樣本送至臨床細胞遺傳學實驗室(估計實驗室至實驗室交通時間為一小時)。在中期(metaphase)分析細胞,此時染色體係最佳可視化。在所計數之二十個處於中期的細胞中,有五個分析出具有正常均質性核型數目(兩個)。如果觀察到兩個核型,即將細胞樣本歸類為均質性。如果觀察到超過兩個核型,即將細胞樣本歸類為異質性。當識別出異質性核型數目(四個)時,即計數額外中期細胞並加以分析。 PPDCs from postnatal tissues of male neonates were cultured in growth medium containing penicillin / streptomycin. Postnatal tissues from male neonates (X, Y) were selected to be able to distinguish neonate-derived cells from maternal-derived cells (X, X). Cells were seeded at 5,000 cells per square centimeter in medium in T25 flasks (Corning Inc., Corning, N.Y.) and expanded to 80% overgrown. Fill the T25 flask containing cells to the neck with growth medium. The courier will send the sample to the clinical cytogenetics laboratory (estimated laboratory to laboratory transportation time is one hour). The cells are analyzed in metaphase, at which time the staining system is best visualized. Of the twenty cells in the metaphase that were counted, five were analyzed for the number of normal homogeneous karyotypes (two). If two karyotypes are observed, the cell sample is classified as homogeneous. If more than two karyotypes are observed, the cell sample is classified as heterogeneous. When the number of heterogeneous karyotypes (four) is identified, additional metaphase cells are counted and analyzed.
所有送交染色體分析的細胞樣本皆判讀為展現正常外觀。16個經分析之細胞系中的三個展現異質性表型(XX與XY),從而指示存在有同時衍生自新生兒與母體來源的細胞(表15-1)。將衍生自組織胎盤-N之細胞自胎盤之新生兒態樣分離。在繼代零, 此細胞系顯現出均質性XY。然而,在繼代九,該細胞系為異質性(XX/XY),指出先前未偵測出的母體來源細胞存在。 All cell samples submitted for chromosome analysis are interpreted as showing normal appearance. Three of the 16 analyzed cell lines exhibited a heterogeneous phenotype (XX and XY), indicating the presence of cells derived from both neonatal and maternal sources (Table 15-1). Cells derived from tissue placenta-N were isolated from the neonatal appearance of the placenta. At passage zero, this cell line exhibited homogeneous XY. However, in generation nine, the cell line was heterogeneous (XX / XY), indicating the presence of maternal-derived cells not previously detected.
總結:染色體分析識別出核型顯現為正常(由臨床細胞遺傳學實驗室判讀)的胎盤及臍衍生細胞。核型分析亦識別出不含母體細胞的細胞系,此係由均質性核型來判定。 Summary: Chromosome analysis identified placenta and umbilical-derived cells whose karyotype appeared normal (interpreted by the clinical cytogenetics laboratory). Karyotype analysis also identified cell lines that did not contain maternal cells, as determined by the homogeneous karyotype.
藉由流動式細胞測量術來表徵細胞表面蛋白質或「標記」可用來判定細胞系的身份(identity)。表現的一致性可由多個捐贈者來判定,並且在暴露於不同處理與培養條件的細胞中判定。自胎盤及臍分離出的產後衍生細胞(PPDC)系係經表徵(藉由流動式細胞測量術),從而提供用於識別這些細胞系的概況。 Characterization of cell surface proteins or "markers" by flow cytometry can be used to determine the identity of cell lines. The consistency of performance can be determined by multiple donors and in cells exposed to different treatment and culture conditions. The postpartum-derived cell (PPDC) lines isolated from the placenta and navel are characterized (by flow cytometry) to provide an overview for identifying these cell lines.
培養基及培養容器:將細胞培養於具有青黴素/鏈黴素之生長培養基(Gibco Carlsbad,Calif.)中。使細胞在經血漿處理之T75、T150與T225組織培養瓶(Corning Inc.,Corning,N.Y.)中培養直到長滿。培養瓶的生長表面係藉由在室溫下培養2%(w/v)明膠(Sigma,St.Louis,Mo.)20分鐘來塗布明膠。 Culture medium and culture vessel: The cells were cultured in a growth medium (Gibco Carlsbad, Calif.) With penicillin / streptomycin. The cells were cultured in plasma-treated T75, T150, and T225 tissue culture flasks (Corning Inc., Corning, N.Y.) until they were overgrown. The growth surface of the culture flask was coated with gelatin by incubating 2% (w / v) gelatin (Sigma, St. Louis, Mo.) at room temperature for 20 minutes.
抗體染色及流動式細胞測量術分析:將瓶中之貼附細胞於PBS中清洗且用胰蛋白酶/EDTA脫附。將細胞收集、離心,且以每毫升1×107的細胞濃度再懸浮於3%(v/v)FBS於PBS中。根據製造商規範,將關注的細胞表面標記之抗體(請見後文)添加至一百微升的細胞懸浮液,然後將混合物在4℃下於黑暗中培養30分鐘。培養之後,將細胞用PBS清洗並離心以移除未結合之抗體。將細胞再懸浮於500微升PBS中且藉由流動式細胞測量術分析。流動式細胞測量術分析係用FACScaliburTM儀器(Becton Dickinson,San Jose,Calif.)來執行。表16-1列舉所使用之細胞表面標記之抗體。 Antibody staining and flow cytometry analysis: The attached cells in the bottle were washed in PBS and desorbed with trypsin / EDTA. The cells were collected, centrifuged, and resuspended in 3% (v / v) FBS in PBS at a cell concentration of 1 × 10 7 per ml. According to the manufacturer's specifications, the antibody labeled on the cell surface of interest (see below) is added to one hundred microliters of the cell suspension, and then the mixture is incubated at 4 ° C in the dark for 30 minutes. After incubation, the cells were washed with PBS and centrifuged to remove unbound antibody. The cells were resuspended in 500 microliters of PBS and analyzed by flow cytometry. Flow cytometry analysis was performed with a FACScalibur ™ instrument (Becton Dickinson, San Jose, Calif.). Table 16-1 lists the cell surface labeled antibodies used.
胎盤與臍比較:在繼代8將胎盤衍生細胞與臍衍生細胞進行比較。 Comparison of placenta and umbilical cord: Placenta-derived cells are compared with umbilical cord-derived cells at the 8th passage.
繼代對繼代比較:在繼代8、15、及20分析胎盤衍生細胞及臍衍生細胞。 Comparison of sub-generation to sub-generation: placenta-derived cells and umbilical-derived cells were analyzed at sub-generations 8, 15, and 20.
捐贈者對捐贈者比較:為了比較捐贈者之間的差異,將來自不同捐贈者之胎盤衍生細胞互相比較,且將來自不同捐贈者之臍衍生細胞互相比較。 Donor-to-donor comparison: To compare the differences between donors, placental-derived cells from different donors are compared with each other, and umbilical-derived cells from different donors are compared with each other.
表面塗層比較:將培養在經明膠塗布之培養瓶上的胎盤衍生細胞與培養於未經塗布之培養瓶上的胎盤衍生細胞進行比較。將培養於經明膠塗布培養瓶上的臍衍生細胞與培養於未塗布培養瓶上之臍衍生細胞進行比較。 Surface coating comparison: compare placental-derived cells cultured on gelatin-coated culture flasks with placenta-derived cells cultured on uncoated culture flasks. Umbilical-derived cells cultured on gelatin-coated culture flasks were compared with umbilical-derived cells cultured on uncoated culture flasks.
消化酶比較:對四種用於分離及製備細胞的處理進行比較。分離自用1)膠原蛋白酶;2)膠原蛋白酶/分散酶;3)膠原蛋白 酶/玻尿酸酶;與4)膠原蛋白酶/玻尿酸酶/分散酶處理之胎盤之細胞係經比較。 Digestive enzyme comparison: compare the four treatments used to isolate and prepare cells. It was isolated from the cell line of placenta treated with 1) collagenase; 2) collagenase / dispase; 3) collagenase / hyaluronidase; and 4) collagenase / hyaluronidase / dispase.
胎盤層比較:將衍生自胎盤組織之母體態樣的細胞與衍生自胎盤組織之絨毛區的細胞以及衍生自胎盤之新生胎兒態樣的細胞進行比較。 Placental layer comparison: Compare the cells derived from the maternal form of placental tissue with the cells derived from the villi region of the placental tissue and the cells derived from the neonatal fetus of the placenta.
胎盤與臍比較:藉由流動式細胞測量術分析之胎盤衍生細胞及臍衍生細胞顯示CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C之陽性表現,此係由相對於IgG對照組的螢光值增加來指示。這些細胞之CD31、CD34、CD45、CD117、CD141、與HLA-DR、HLA-DP、HLA-DQ的可偵測表現為陰性,此係由螢光值與IgG對照組可相比來指示。考慮到陽性曲線之螢光值之變異。陽性曲線之平均值(亦即CD13)及範圍(亦即CD90)顯示一些變異,但曲線顯現正常,證實為均質性族群。兩個曲線個別展現大於IgG對照組之值。 Comparison of placenta and umbilicus: Placenta-derived cells and umbilical-derived cells analyzed by flow cytometry showed positive expression of CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA-B, HLA-C This is indicated by the increase in fluorescence value relative to the IgG control group. The detectable performance of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, and HLA-DQ of these cells is negative, which is indicated by the comparison of the fluorescence value with the IgG control group. Consider the variation of the fluorescence value of the positive curve. The mean (ie CD13) and range (ie CD90) of the positive curve showed some variation, but the curve appeared normal, confirming a homogeneous population. The two curves individually show values greater than the IgG control group.
繼代對繼代比較-胎盤衍生細胞:藉由流動式細胞測量術分析之繼代8、15、及20的胎盤衍生細胞皆陽性表現CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C,如相對於IgG對照組的螢光值增加所反映。該些細胞之CD31、CD34、CD45、CD117、CD141與HLA-DR、HLA-DP、HLA-DQ的表現為陰性,具有與IgG對照組一致的螢光值。 Passage-to-passage comparison-placental-derived cells: placental-derived cells analyzed by flow cytometry at passage 8, 15, and 20 were positive for CD10, CD13, CD44, CD73, CD90, PDGFr-α, and HLA -A, HLA-B, HLA-C, as reflected by the increase in fluorescence value relative to the IgG control group. The CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, and HLA-DQ of these cells were negative, and had the same fluorescence value as the IgG control group.
繼代對繼代比較-臍衍生細胞:藉由流動式細胞測量術分析之繼代8、15、及20之臍衍生細胞皆表現CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C,此係由相對於IgG對照組的螢光增加來指示。這些細胞為對CD31、CD34、CD45、CD117、CD141與HLA-DR、HLA-DP、HLA-DQ陰性,此係由螢光值與IgG對照組一致來指示。 Passage to Passage Comparison-Umbilical Derived Cells: Umbilical derived cells analyzed by flow cytometry at passage 8, 15, and 20 all express CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA- A, HLA-B, HLA-C, this is indicated by the increase in fluorescence relative to the IgG control group. These cells are negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, HLA-DQ, which is indicated by the fluorescence value consistent with the IgG control group.
捐贈者對捐贈者比較-胎盤衍生細胞:藉由流動式細胞測量術分析分離自不同捐贈者之胎盤衍生細胞各表現CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C,其相對於IgG對照組具有增加的螢光值。該些細胞為CD31、CD34、CD45、CD117、CD141與HLA-DR、HLA-DP、HLA-DQ表現陰性,此係由螢光值與IgG對照組一致來指示。 Donor-to-donor comparison-placenta-derived cells: analysis of the expression of CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA- by placental-derived cells isolated from different donors by flow cytometry B. HLA-C, which has an increased fluorescence value relative to the IgG control group. These cells were CD31, CD34, CD45, CD117, CD141 and negative for HLA-DR, HLA-DP, HLA-DQ, which is indicated by the fluorescence value consistent with the IgG control group.
捐贈者對捐贈者比較-臍衍生細胞:藉由流動式細胞測量術分析分離自不同捐贈者之臍衍生細胞各顯示CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C之陽性表現,此係反映在相對於IgG對照組增加的螢光值。這些細胞之CD31、CD34、CD45、CD117、CD141與HLA-DR、HLA-DP、HLA-DQ的表現為陰性,具有與IgG對照組一致的螢光值。 Donor-to-donor comparison-umbilical derived cells: analysis of umbilical derived cells isolated from different donors by flow cytometry showed CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA- B. The positive performance of HLA-C is reflected in the increased fluorescence value relative to the IgG control group. The expression of CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, and HLA-DQ of these cells were negative, and they had the same fluorescence value as the IgG control group.
明膠表面塗層對胎盤衍生細胞的效果:藉由流動式細胞測量術分析在經明膠塗布或未經塗布之培養瓶上擴增的胎盤衍生細胞皆表現CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C,此係反映在相對於IgG對照組增加的螢光值。這 些細胞為CD31、CD34、CD45、CD117、CD141與HLA-DR、HLA-DP、HLA-DQ表現陰性,此係由螢光值與IgG對照組一致來指示。 The effect of gelatin surface coating on placenta-derived cells: analysis of placenta-derived cells expanded on gelatin-coated or uncoated culture flasks by flow cytometry showed CD10, CD13, CD44, CD73, CD90, PDGFr -α and HLA-A, HLA-B, HLA-C, this is reflected in the increased fluorescence value relative to the IgG control group. These cells were CD31, CD34, CD45, CD117, CD141 and negative for HLA-DR, HLA-DP, HLA-DQ, which is indicated by the fluorescence value consistent with the IgG control group.
明膠表面塗層對臍衍生細胞的效果:藉由流動式細胞測量術分析在明膠上及在未經塗布之培養瓶上擴增的臍衍生細胞皆陽性表現CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C,其相對於IgG對照組具有增加的螢光值。這些細胞之CD31、CD34、CD45、CD117、CD141與HLA-DR、HLA-DP、HLA-DQ的表現為陰性,具有與IgG對照組一致的螢光值。 Effect of gelatin surface coating on umbilical-derived cells: analysis of umbilical-derived cells expanded on gelatin and uncoated culture flasks by flow cytometry showed positive expression CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA-B, and HLA-C have increased fluorescence values relative to the IgG control group. The expression of CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, and HLA-DQ of these cells were negative, and they had the same fluorescence value as the IgG control group.
用於製備細胞之酶消化程序對細胞表面標記概況的效果:藉由流動式細胞測量術分析之使用各種消化酶分離的胎盤衍生細胞皆表現CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C,如藉由相對於IgG對照組之螢光值增加所指示。這些細胞為CD31、CD34、CD45、CD117、CD141與HLA-DR、HLA-DP、HLA-DQ表現陰性,如藉由螢光值與IgG對照組一致來指示。 Effect of enzymatic digestion procedures used to prepare cells on cell surface marker profiles: Placenta-derived cells isolated using various digestive enzymes analyzed by flow cytometry showed CD10, CD13, CD44, CD73, CD90, PDGFr-α and HLA-A, HLA-B, HLA-C, as indicated by the increase in fluorescence value relative to the IgG control group. These cells were negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, HLA-DQ, as indicated by the fluorescence values consistent with the IgG control group.
胎盤層比較:藉由流動式細胞測量術分析之分別分離自胎盤之母體、絨毛、及新生兒層的細胞顯示CD10、CD13、CD44、CD73、CD90、PDGFr-α及HLA-A、HLA-B、HLA-C之陽性表現,如藉由相對於IgG對照組之螢光值增加所指示。這些細胞為CD31、CD34、CD45、CD117、CD141與HLA-DR、HLA-DP、HLA-DQ表現陰性,如藉由螢光值與IgG對照組一致來指示。 Placental layer comparison: Cells isolated from the maternal, villi, and neonatal layers of the placenta analyzed by flow cytometry showed CD10, CD13, CD44, CD73, CD90, PDGFr-α, and HLA-A, HLA-B , The positive performance of HLA-C, as indicated by the increase in fluorescence value relative to the IgG control group. These cells were negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, HLA-DP, HLA-DQ, as indicated by the fluorescence values consistent with the IgG control group.
總結:胎盤衍生細胞及臍衍生細胞的流動式細胞測量術分析已建立這些細胞系的身份。胎盤衍生細胞及臍衍生細胞為對CD10、CD13、CD44、CD73、CD90、PDGFr-α、HLA-A、HLA-B、HLA-C陽性,且為對CD31、CD34、CD45、CD117、CD141及HLA-DR、HLA-DP、HLA-DQ陰性。此身份在變項包括捐贈者、繼代、培養容器表面塗層、消化酶與胎盤層的變異間皆一致。觀察到在個別螢光值直方圖曲線平均值與範圍上有一些變異,但在所有測試條件下的所有陽性曲線皆為正常並且表現出大於IgG對照組的螢光值,因而確認該些細胞包含具有該些標記之陽性表現的均質性族群。 Summary: Flow cytometry analysis of placenta-derived cells and umbilical-derived cells has established the identity of these cell lines. Placenta-derived cells and umbilical-derived cells are positive for CD10, CD13, CD44, CD73, CD90, PDGFr-α, HLA-A, HLA-B, HLA-C, and are positive for CD31, CD34, CD45, CD117, CD141 and HLA -DR, HLA-DP, HLA-DQ negative. This identity is consistent among the variables including donor, sub-generation, culture vessel surface coating, digestive enzymes, and placental layer variation. Some variation in the average and range of individual fluorescence value histogram curves was observed, but all positive curves under all test conditions were normal and exhibited fluorescence values greater than the IgG control group, thus confirming that these cells contained A homogeneous group with positive expressions of these markers.
人類產後組織(即臍帶及胎盤)中所發現的細胞之表型係以免疫組織化學法分析。 The phenotypes of cells found in human postpartum tissues (ie, umbilical cord and placenta) were analyzed by immunohistochemistry.
組織製備:將人類臍帶及胎盤組織收集,並在4℃下浸漬固定於4%(w/v)多聚甲醛中過夜。使用針對下列表位的抗體來執行免疫組織化學法:波形蛋白(1:500;Sigma,St.Louis,Mo.)、肌間線蛋白(desmin,1:150,對抗兔而生成;Sigma;或1:300,對抗小鼠而生成;Chemic on,Temecula,Calif.),α-平滑肌肌動蛋白(SMA; 1:400;Sigma),細胞角蛋白18(CK18;1:400;Sigma),馮威里氏因子(vWF;1:200;Sigma)與CD34(人類CD34 Class III;1:100;DAKOCytomation,Carpinteria,Calif)。此外,測試以下標記:抗人類GROα--PE(1:100;Becton Dickinson,Franklin Lakes,N.J.)、抗人類GCP-2(1:100;Santa Cruz Biotech,Santa Cruz,Calif)、抗人類氧化LDL受體1(ox-LDL R1;1:100;Santa Cruz Biotech)與抗人類NOGO-A(1:100;Santa Cruz Biotech)。用解剖刀修剪經固定之樣品,然後將其置於在含乙醇之乾冰浴上的OCT包埋化合物(Tissue-Tek OCT;Sakura,Torrance,Calif)內。接著使用標準低溫恒溫器(Leica Microsystems)將冷凍之包埋塊切片(10μm厚)然後固定於玻片上以備染色。 Tissue preparation: Human umbilical cord and placental tissues were collected and immersed and fixed in 4% (w / v) paraformaldehyde at 4 ° C overnight. Immunohistochemistry was performed using antibodies against the following epitopes: vimentin (1: 500; Sigma, St. Louis, Mo.), intermycin (desmin, 1: 150, generated against rabbits; Sigma; or 1: 300, generated against mice; Chemic on, Temecula, Calif.), Α-smooth muscle actin (SMA; 1: 400; Sigma), cytokeratin 18 (CK18; 1: 400; Sigma), Feng Weili Factor (vWF; 1: 200; Sigma) and CD34 (human CD34 Class III; 1: 100; DAKOCytomation, Carpinteria, Calif). In addition, the following markers were tested: anti-human GROα-PE (1: 100; Becton Dickinson, Franklin Lakes, NJ), anti-human GCP-2 (1: 100; Santa Cruz Biotech, Santa Cruz, Calif), anti-human oxidized LDL Receptor 1 (ox-LDL R1; 1: 100; Santa Cruz Biotech) and anti-human NOGO-A (1: 100; Santa Cruz Biotech). The fixed sample was trimmed with a scalpel and then placed in an OCT embedding compound (Tissue-Tek OCT; Sakura, Torrance, Calif) on a dry ice bath containing ethanol. Next, the frozen embedded block was sliced (10 μm thick) using a standard cryostat (Leica Microsystems) and then fixed on a glass slide for staining.
免疫組織化學:類似於先前研究執行免疫組織化學(例如,Messina等人,2003,Exper.Neurol.184:816-829)。用磷酸鹽緩衝液(PBS)清洗組織切片然後將其暴露於含PBS、4%(v/v)山羊血清(Chemic on,Temecula,Calif)與0.3%(v/v)Triton(Triton X-100;Sigma)的蛋白質阻斷液中1小時以獲取細胞內抗原。在感興趣表位會位於細胞表面(CD34、ox-LDL R1)上的情況中,省略該程序的所有步驟中之Triton以避免表位流失。再者,在一級抗體係對抗山羊(GCP-2,ox-LDL R1,NOGO-A)而生成的情況中,整個程序中使用3%(v/v)驢血清來取代山羊血清。接著在室溫下將一級抗體(稀釋於阻斷液中)施用於切片歷時4小時。將一級抗體溶液移除,用PBS清洗培養物,然後施用二級抗體溶液(在室溫下1小時),該二級抗 體溶液含有阻斷劑以及山羊抗小鼠IgG--Texas Red(1:250;Molecular Probes,Eugene,Oreg.)及/或山羊抗兔IgG-Alexa 488(1:250;Molecular Probes)或驢抗山羊IgG--FITC(1:150;Santa Cruz Biotech)。清洗培養物,然後施用10微莫耳DAPI(Molecular Probes)10分鐘以可視化細胞核。 Immunohistochemistry: Immunohistochemistry is performed similar to previous studies (for example, Messina et al., 2003, Exper. Neurol. 184: 816-829). Wash tissue sections with phosphate buffered saline (PBS) and then expose them to PBS, 4% (v / v) goat serum (Chemic on, Temecula, Calif) and 0.3% (v / v) Triton (Triton X-100 ; Sigma) protein blocking solution for 1 hour to obtain intracellular antigen. In the case where the epitope of interest will be located on the cell surface (CD34, ox-LDL R1), Triton is omitted in all steps of the procedure to avoid epitope loss. Furthermore, in the case where the primary antibody system was generated against goats (GCP-2, ox-LDL R1, NOGO-A), 3% (v / v) donkey serum was used in place of goat serum in the entire procedure. Next, primary antibodies (diluted in blocking solution) were applied to the slices at room temperature for 4 hours. The primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking agent and goat anti-mouse IgG--Texas Red (1: 250; Molecular Probes, Eugene, Oreg.) And / or goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes) or donkey anti-goat IgG-FITC (1: 150; Santa Cruz Biotech). The culture was washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.
在免疫染色後,在Olympus倒立落射螢光顯微鏡(Olympus,Melville,N.Y.)上使用適當螢光濾光片來可視化螢光。高於對照組染色的螢光信號即代表陽性染色。使用數位彩色攝影機與ImagePro軟體(Media Cybernetics,Carlsbad,Calif.)來擷取代表影像。針對三重染色的樣本,一次僅使用一個發射濾光片來拍攝各影像。接著使用Adobe Photoshop軟體(Adobe,San Jose,Calif.)來製備分層合成影像(Layered montage)。 After immunostaining, the fluorescence is visualized using an appropriate fluorescent filter on an Olympus inverted epifluorescence microscope (Olympus, Melville, N.Y.). The fluorescent signal higher than the staining of the control group represents positive staining. Use a digital color camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.) To capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop software (Adobe, San Jose, Calif.) To prepare layered composite images (Layered montage).
臍帶之表徵:波形蛋白、肌間線蛋白、SMA、CK18、vWF、及CD34標記係表現於臍帶內發現之細胞亞群中。特定而言,vWF與CD34表現限制於臍帶內所含之血管。CD34+細胞係位於最內層(管腔側)上。波形蛋白表現係發現於整個臍帶基質與血管中。SMA限於動脈及靜脈的基質與外壁,但血管本身則不含。僅在血管內觀察到CK18與肌間線蛋白,且肌間線蛋白限於中層及外層。 Characterization of the umbilical cord: Vimentin, intermuscular fibroin, SMA, CK18, vWF, and CD34 markers are expressed in the subpopulations of cells found in the umbilical cord. Specifically, the performance of vWF and CD34 is limited to the blood vessels contained in the umbilical cord. The CD34 + cell line is located on the innermost layer (lumen side). Vimentin expression is found throughout the umbilical cord matrix and blood vessels. SMA is limited to the matrix and outer wall of arteries and veins, but the blood vessels themselves are not. CK18 and intermuscular fibroin were only observed in blood vessels, and intermuscular fibroin was limited to the middle and outer layers.
胎盤之表徵:波形蛋白、肌間線蛋白、SMA、CK18、vWF、及CD34全部在胎盤內觀察到且為區域特異性。 Characterization of the placenta: vimentin, intermuscular protein, SMA, CK18, vWF, and CD34 are all observed in the placenta and are region-specific.
GROα、GCP-2、ox-LDL RI、及NOGO-A組織表現:此等標記皆未在臍帶或胎盤組織內觀察到。 GROα , GCP-2 , ox-LDL RI , and NOGO-A tissue performance: none of these markers were observed in the umbilical cord or placental tissue.
總結:人類臍帶及胎盤內的細胞中皆會表現波形蛋白、肌間線蛋白、α-平滑肌肌動蛋白、細胞角蛋白18、馮威里氏因子、及CD 34。 Summary: Cells in the human umbilical cord and placenta exhibit vimentin, intermuscular fibroin, α-smooth muscle actin, cytokeratin 18, von Willie's factor, and CD 34.
Affymetrix GENECHIP陣列係用來比較臍衍生與胎盤衍生細胞與纖維母細胞、人類間葉幹細胞與另一個衍生自人類骨髓之細胞系的基因表現概況。此分析提供產後衍生細胞之特徵並識別這些細胞的獨特分子標記。 The Affymetrix GENECHIP array is used to compare gene expression profiles of umbilical-derived and placental-derived cells and fibroblasts, human mesenchymal stem cells, and another cell line derived from human bone marrow. This analysis provides the characteristics of postpartum-derived cells and unique molecular markers that identify these cells.
細胞之分離及培養:人類臍帶與胎盤係獲自國家疾病研究交換中心(NDRI,Philadelphia,Pa.),其等係來自正常足月分娩並且獲得患者同意。如實例14中所述接受組織並分離細胞。將細胞培養於經明膠塗布的組織培養塑膠培養瓶上之生長培養基(使用DMEM-LG)中。培養物係在37℃下以5% CO2培養。 Isolation and cultivation of cells: The human umbilical cord and placenta were obtained from the National Center for Disease Research and Exchange (NDRI, Philadelphia, Pa.), Which was derived from normal full-term delivery and obtained patient consent. The tissue was received and the cells were isolated as described in Example 14. Cells were cultured in growth medium (using DMEM-LG) on gelatin-coated tissue culture plastic culture flasks. The culture line was cultured at 37 ° C with 5% CO 2 .
人類皮膚纖維母細胞係購自Cambrex Incorporated(Walkersville,Md.;批號9F0844)與ATCC CRL-1501(CCD39SK)。將兩個細胞系在DMEM/F12培養基(Invitrogen,Carlsbad,Calif.)中培 養,培養基含有10%(v/v)胎牛血清(Hyclone)與青黴素/鏈黴素(Invitrogen)。使細胞生長於標準組織處理塑膠上。 Human skin fibroblast cell lines were purchased from Cambrex Incorporated (Walkersville, Md .; batch number 9F0844) and ATCC CRL-1501 (CCD39SK). The two cell lines were cultured in DMEM / F12 medium (Invitrogen, Carlsbad, Calif.) Containing 10% (v / v) fetal bovine serum (Hyclone) and penicillin / streptomycin (Invitrogen). Allow cells to grow on standard tissue processing plastic.
人類間葉幹細胞(hMSC)係購自Cambrex Incorporated(Walkersville,Md.;批號2F1655、2F1656與2F1657)並根據製造商規範在MSCGM培養基(Cambrex)中培養。使細胞在37℃下以5% CO2生長於標準組織培養塑膠上。 Human mesenchymal stem cells (hMSC) were purchased from Cambrex Incorporated (Walkersville, Md .; batch numbers 2F1655, 2F1656 and 2F1657) and cultured in MSCGM medium (Cambrex) according to the manufacturer's specifications. The cells were grown on standard tissue culture plastics at 37 ° C with 5% CO 2 .
人類髂骨崤骨髓係接受自NDRI並獲得患者同意。骨髓係根據Ho等人(W003/025149)所概述之方法來處理。將骨髓與裂解緩衝液(155mM NH4Cl、10mM KHCO3與0.1mM EDTA,pH 7.2)混合,並且混合比例為1份骨髓對20份裂解緩衝液。使細胞懸浮液渦動、在環境溫度下培養2分鐘然後以500xg離心10分鐘。將上清液丟棄,並將細胞團塊再懸浮於補充10%(v/v)胎牛血清與4mM麩醯胺酸之最低必需培養基α(Invitrogen)中。將細胞再次離心然後將細胞團塊再懸浮於新鮮培養基中。使用台盼藍排除法(Sigma,St.Louis,Mo.)來計數存活單核細胞。將單核細胞以5×104個細胞/cm2接種於組織培養塑膠瓶中。將細胞在37℃下以5% CO2、在標準大氣O2或在5% O2下培養。將細胞培養5日並且未進行培養基更換。在5日的培養後將培養基與非貼附細胞移除。將貼附細胞維持於培養中。 The human iliac bone marrow system was received from NDRI and obtained patient consent. Bone marrow is processed according to the method outlined by Ho et al. (W003 / 025149). Bone marrow was mixed with lysis buffer (155 mM NH4Cl, 10 mM KHCO 3 and 0.1 mM EDTA, pH 7.2), and the mixing ratio was 1 part bone marrow to 20 parts lysis buffer. The cell suspension was vortexed, incubated at ambient temperature for 2 minutes and then centrifuged at 500 x g for 10 minutes. The supernatant was discarded, and the cell pellet was resuspended in the minimum essential medium α (Invitrogen) supplemented with 10% (v / v) fetal bovine serum and 4 mM glutamic acid. The cells were centrifuged again and the cell pellet was resuspended in fresh medium. The trypan blue exclusion method (Sigma, St. Louis, Mo.) was used to count viable monocytes. Mononuclear cells were seeded in tissue culture plastic bottles at 5 × 10 4 cells / cm 2 . The cells were cultured at 37 ° C with 5% CO 2 , standard atmospheric O 2, or 5% O 2 . The cells were cultured for 5 days without medium replacement. After 5 days of cultivation, the medium and non-attached cells were removed. The adherent cells are maintained in culture.
mRNA之分離及GENECHIP分析:將活性生長之細胞培養物用細胞刮勺自培養瓶移除於冷PBS中。將細胞以300xg離心5分鐘。將上清液移除,然後將細胞再懸浮於新鮮PBS並再次離心。 將上清液移除,然後立即將細胞團塊冷凍並儲存在-80℃下。將細胞mRNA萃取出來然後轉錄為cDNA,隨後將其轉錄成cRNA且經生物素標記。使經生物素標記之cRNA與HG-U133A GENECHIP寡核苷酸陣列(Affymetrix,Santa Clara,Calif.)雜交。雜交與數據收集係根據製造商規範來執行。分析係使用「Significance Analysis of Microarrays」(SAM)第1.21版電腦軟體(Stanford University;Tusher,V.G.等人,2001,Proc.Natl.Acad.Sci.USA 98:5116-5121)執行。 Isolation of mRNA and GENECHIP analysis: Actively growing cell culture was removed from the culture flask in cold PBS using a cell spatula. Centrifuge the cells at 300 x g for 5 minutes. The supernatant was removed, then the cells were resuspended in fresh PBS and centrifuged again. The supernatant was removed, and immediately the cell pellet was frozen and stored at -80 ° C. Cell mRNA is extracted and transcribed into cDNA, which is then transcribed into cRNA and labeled with biotin. The biotin-labeled cRNA was hybridized to the HG-U133A GENECHIP oligonucleotide array (Affymetrix, Santa Clara, Calif.). Hybridization and data collection are performed according to the manufacturer's specifications. The analysis was performed using "Significance Analysis of Microarrays" (SAM) version 1.21 computer software (Stanford University; Tusher, VG et al., 2001, Proc. Natl. Acad. Sci. USA 98: 5116-5121).
分析了十四個不同細胞群。細胞以及繼代資訊、培養基材與培養基係列於表18-1。 Fourteen different cell populations were analyzed. Table 18-1 lists the cells and information on the subcultures, culture media and culture medium series.
數據藉由主組分分析(Principle Component Analysis)評估,分析在細胞中有差別表現的290個基因。此分析允許族群之間相似性之相對比較。 The data was evaluated by Principle Component Analysis, which analyzed 290 genes with differential performance in cells. This analysis allows relative comparison of similarities between ethnic groups.
表18-2顯示計算用於細胞對比較之歐幾里德距離(Euclidean distance)。歐幾里德距離係根據基於290個基因的細胞比較結果,這些基因在不同細胞型間有差別表現。歐幾里德距離與290個基因之表現之間的相似性成反比(亦即,距離越大,存在的相似性越小)。 Table 18-2 shows the calculation of Euclidean distance for cell pair comparison. The Euclidean distance is based on the comparison of cells based on 290 genes, which have different performances among different cell types. The Euclidean distance is inversely proportional to the similarity between the performance of 290 genes (that is, the greater the distance, the less similarity exists).
表18-3、18-4與18-5顯示在胎盤衍生細胞中增加表現(表18-3)、在臍衍生細胞中增加表現(表18-4)及在臍與胎盤衍生細胞中減少表現(表18-5)之基因。標題「探針組ID」之欄係指製造商對於定位於晶片上具體位點上之數種寡核苷酸探針組之識別碼,該些探針組與經列名之基因(「基因名稱」欄)雜交,該等基 因包含可在NCBI(GenBank)數據庫內以指定存取號(「NCBI存取號」欄)查找之序列。 Tables 18-3, 18-4, and 18-5 show increased performance in placenta-derived cells (Table 18-3), increased performance in umbilical-derived cells (Table 18-4), and decreased performance in umbilical and placenta-derived cells (Table 18-5) Genes. The column titled "Probe Set ID" refers to the manufacturer's identification codes for several oligonucleotide probe sets located at specific locations on the chip. These probe sets and the listed genes ("genes "Name" column) hybridization. These genes contain sequences that can be found in the NCBI (GenBank) database with the specified access number ("NCBI Access Number" column).
表18-6、18-7與18-8顯示在人類纖維母細胞(表18-6)、ICBM細胞(表18-7)與MSC(表18-8)中增加表現之基因。 Tables 18-6, 18-7, and 18-8 show genes that increase expression in human fibroblasts (Table 18-6), ICBM cells (Table 18-7), and MSC (Table 18-8).
總結:本檢測係為了提供衍生自臍帶及胎盤之產後細胞的分子表徵而執行。此分析包括衍生自三個不同臍帶與三個不同胎盤的細胞。此檢測亦包括兩個不同的皮膚纖維母細胞系、三個間葉幹細胞系與三個髂骨崤骨髓細胞系。這些細胞所表現之mRNA係使用寡核苷酸陣列來分析,該陣列含有22,000個基因的探針。結果 顯示,290個基因在這五種不同細胞型中有差別表現。這些基因包括十個在胎盤衍生細胞中特異地增加的基因,以及七個在臍帶衍生細胞中特異地增加的基因。發現相較於其他細胞型,四十四個基因具有在胎盤與臍帶中特異地較低的表現水準。選定基因之表現已藉由PCR來確認(參見以下實例)。此等結果證明,產後衍生細胞具有獨特的基因表現概況,例如相較於骨髓衍生細胞及纖維母細胞。 Summary: This test was performed to provide molecular characterization of postnatal cells derived from the umbilical cord and placenta. This analysis included cells derived from three different umbilical cords and three different placenta. The test also included two different skin fibroblast cell lines, three mesenchymal stem cell lines, and three iliac bone marrow cell lines. The mRNA expressed by these cells was analyzed using an oligonucleotide array containing probes of 22,000 genes. The results showed that 290 genes performed differently in these five different cell types. These genes include ten genes that are specifically increased in placenta-derived cells, and seven genes that are specifically increased in umbilical cord-derived cells. It was found that forty-four genes have specifically lower performance levels in the placenta and umbilical cord compared to other cell types. The performance of selected genes has been confirmed by PCR (see example below). These results prove that postpartum-derived cells have a unique gene expression profile, for example, compared to bone marrow-derived cells and fibroblasts.
在前述實例中,衍生自人類胎盤及人類臍帶之細胞的相似性及差異性係藉由比較其基因表現概況與衍生自其他來源之細胞的基因表現概況來評估(使用寡核苷酸陣列)。識別六個「簽名(signature)」基因:氧化LDL受體1、介白素-8、凝乳酶、內質網蛋白、趨化激素受體配體3(CXC配體3)、及顆粒性細胞趨化蛋白質2(GCP-2)。此等「簽名」基因在產後衍生細胞中以相對高的水準表現。 In the foregoing examples, the similarity and difference of cells derived from human placenta and human umbilical cord were evaluated by comparing their gene performance profiles with those of cells derived from other sources (using oligonucleotide arrays). Identify six "signature" genes: oxidized LDL receptor 1, interleukin-8, rennet, endoplasmic reticulum protein, chemokine receptor ligand 3 (CXC ligand 3), and granularity Cell chemotactic protein 2 (GCP-2). These "signature" genes are expressed at a relatively high level in postpartum derived cells.
進行此實例中所述之程序以驗證微陣列數據且找出基因與蛋白質表現之間的一致性/不一致性,以及建立一系列可靠的檢定以用於偵測胎盤衍生細胞與臍衍生細胞之唯一識別符。 Perform the procedure described in this example to verify the microarray data and find the consistency / inconsistency between gene and protein performance, and establish a series of reliable tests for detecting the uniqueness of placenta-derived cells and umbilical-derived cells Identifier.
細胞:胎盤衍生細胞(三個分離株,包括一個經核型分析所識別主要為新生兒之分離株)、臍衍生細胞(四個分離株)、及正常人類皮膚纖維母細胞(NHDF;新生兒及成人),生長於經明膠塗布之T75瓶中具有青黴素/鏈黴素之生長培養基中。間葉幹細胞(MSCS)係生長於間葉幹細胞生長培養基Bullet套組(MSCGM;Cambrex,Walkerville,Md.)。 Cells: Placenta-derived cells (three isolates, including one isolate identified by karyotyping as mainly neonates), umbilical-derived cells (four isolates), and normal human skin fibroblasts (NHDF; neonates And adults), grown in growth medium with penicillin / streptomycin in T75 bottles coated with gelatin. Mesenchymal stem cells (MSCS) lines were grown on the Mesothelial stem cell growth medium Bullet set (MSCGM; Cambrex, Walkerville, Md.).
至於IL-8規程,將細胞自液態氮解凍並以5,000個細胞/cm2接種於經明膠塗布之瓶中,於生長培養基中生長48小時且隨後於10毫升的血清饑餓培養基[DMEM--低葡萄糖(Gibco,Carlsbad,Calif.)、青黴素/鏈黴素(Gibco,Carlsbad,Calif.)及0.1%(w/v)牛血清白蛋白(BSA;Sigma,St.Louis,Mo.)]中再生長8小時。在此處理之後,萃取RNA且將上清液以150xg離心5分鐘以移除細胞碎屑。隨後將上清液冷凍在-80℃下以供ELISA分析。 As for the IL-8 protocol, cells were thawed from liquid nitrogen and seeded in gelatin-coated bottles at 5,000 cells / cm 2 , grown in growth medium for 48 hours and then in 10 ml of serum starvation medium [DMEM-low Glucose (Gibco, Carlsbad, Calif.), Penicillin / streptomycin (Gibco, Carlsbad, Calif.) And 0.1% (w / v) bovine serum albumin (BSA; Sigma, St. Louis, Mo.)) Grow for 8 hours. After this treatment, RNA was extracted and the supernatant was centrifuged at 150 x g for 5 minutes to remove cell debris. The supernatant was then frozen at -80 ° C for ELISA analysis.
ELISA檢定之細胞培養物:將衍生自胎盤及臍之產後細胞、以及衍生自人類新生兒包皮之人類纖維母細胞培養在經明膠塗布之T75培養瓶中的生長培養基中。將細胞在繼代11時冷凍於液態氮中。將細胞解凍且轉移至15毫升離心管。在以150xg離心5分鐘之後,將上清液丟棄。將細胞再懸浮於4毫升培養基中且計數。使細胞以375,000個細胞/培養瓶生長於含有15毫升生長培養基之75cm2培養瓶中24小時。將培養基更換成血清饑餓培養基8小時。在培養結束時以14,000xg離心5分鐘來收集血清饑餓培養基(且保存在-20℃下)。 ELISA- tested cell culture: Post-natal cells derived from placenta and umbilicus, and human fibroblasts derived from human neonatal foreskin are cultured in growth medium in gelatin-coated T75 culture flasks. The cells were frozen in liquid nitrogen at 11th passage. Thaw the cells and transfer to a 15 ml centrifuge tube. After centrifugation at 150 x g for 5 minutes, the supernatant was discarded. The cells were resuspended in 4 ml of medium and counted. The cells were grown at 375,000 cells / flask in a 75 cm 2 flask containing 15 ml of growth medium for 24 hours. The medium was replaced with serum starvation medium for 8 hours. At the end of the culture, centrifuge at 14,000 x g for 5 minutes to collect serum starvation medium (and stored at -20 ° C).
為了估計各培養瓶中細胞之數目,各培養瓶中添加2毫升胰蛋白酶/EDTA(Gibco,Carlsbad,Calif)。在細胞自培養瓶脫離之後,用8毫升的生長培養基中和胰蛋白酶活性。將細胞轉移至15毫升離心管且以150xg離心5分鐘。移除上清液且將1毫升生長培養基添加至各管以再懸浮細胞。使用血球計估計細胞數目。 In order to estimate the number of cells in each flask, 2 mL of trypsin / EDTA (Gibco, Carlsbad, Calif) was added to each flask. After the cells were detached from the culture flask, trypsin activity was neutralized with 8 ml of growth medium. Transfer cells to a 15 ml centrifuge tube and centrifuge at 150 x g for 5 minutes. The supernatant was removed and 1 ml of growth medium was added to each tube to resuspend the cells. Estimate the cell number using a hemocytometer.
ELISA檢定:由細胞分泌至血清饑餓培養基的IL-8之量係使用ELISA檢定(R&D Systems,Minneapolis,Minn.)來分析。所有檢定皆根據製造商所提供之說明書來測試。 ELISA assay: The amount of IL-8 secreted from cells to serum starvation medium was analyzed using ELISA assay (R & D Systems, Minneapolis, Minn.). All verifications are tested according to the instructions provided by the manufacturer.
總RNA分離:將RNA自長滿之產後衍生細胞及纖維母細胞萃取出、或就IL-8表現而言自如上文所述處理之細胞萃取。將細胞用350微升含有β-巰基乙醇(Sigma,St.Louis,Mo.)之緩衝液RLT溶解,此係根據製造商之說明書進行(RNeasy® Mini Kit;Qiagen,Valencia,Calif)。RNA係根據製造商之說明書萃取(RNeasy® Mini Kit;Qiagen,Valencia,Calif),並使其進行DNase處理(2.7U/樣本)(Sigma St.Louis,Mo.)。用50微升經DEPC處理水將RNA沖提出來然後儲存於-80℃下。 Total RNA isolation: RNA is extracted from overgrown postpartum-derived cells and fibroblasts, or in terms of IL-8 performance from cells treated as described above. The cells were lysed with 350 μl of buffer RLT containing β-mercaptoethanol (Sigma, St. Louis, Mo.) according to the manufacturer's instructions (RNeasy ® Mini Kit; Qiagen, Valencia, Calif). RNA was extracted according to the manufacturer's instructions (RNeasy ® Mini Kit; Qiagen, Valencia, Calif) and subjected to DNase treatment (2.7 U / sample) (Sigma St. Louis, Mo.). RNA was extracted with 50 microliters of DEPC-treated water and stored at -80 ° C.
反轉錄:RNA亦自人類胎盤及臍萃取出。將組織(30毫克)懸浮於700微升含有2-巰基乙醇之緩衝液RLT中。將樣本機械均質化且根據製造商之規範進行RNA萃取。用50微升經DEPC處理水將RNA萃取出來然後儲存於-80℃下。使用隨機六聚物以TaqMan®反轉錄試劑(Applied Biosystems,Foster City,Calif.)來反轉 錄RNA,反轉錄在25℃下歷時10分鐘、在37℃下歷時60分鐘、且在95℃下歷時10分鐘。樣本儲存於-20℃。 Reverse transcription: RNA is also extracted from the human placenta and navel. The tissue (30 mg) was suspended in 700 microliters of 2-mercaptoethanol buffer RLT. The samples were mechanically homogenized and RNA extraction was performed according to the manufacturer's specifications. RNA was extracted with 50 microliters of DEPC-treated water and stored at -80 ° C. Using random hexamers reverse transcription reagents to TaqMan ® (Applied Biosystems, Foster City, Calif .) By reverse transcription of RNA, anti-transcribed over at 25 ℃ 10 minutes duration at 37 ℃ 60 minutes and at 95 deg.] C over 10 minutes. Store samples at -20 ° C.
藉由cDNA微陣列識別為在產後細胞中所獨特調節之基因(簽名基因--包括氧化LDL受體、介白素-8、凝乳酶、及內質網蛋白)係使用即時及傳統PCR進一步研究。 Identification of genes uniquely regulated in postpartum cells by cDNA microarrays (signature genes--including oxidized LDL receptors, interleukin-8, rennet, and endoplasmic reticulum proteins) was further performed using real-time and traditional PCR the study.
即時PCR:使用Assays-on-Demand®基因表現產物對cDNA樣本執行PCR:使用具有ABI Prism 7000 SDS軟體之7000序列偵測系統(Applied Biosystems,Foster City,Calif.),根據製造商之說明書(Applied Biosystems,Foster City,Calif.),將氧化LDL受體(Hs00234028);凝乳酶(Hs00166915);內質網蛋白(Hs003825 15);CXC配體3(Hs00171061);GCP-2(Hs00605742);IL-8(Hs00174103);及GAPDH(Applied Biosystems,Foster City,Calif.)與cDNA及TaqMan® Universal PCR主混合物混合。熱循環條件為開始50℃歷時2min及95℃歷時10min,接著為40個95℃歷時15sec及60℃歷時1min之循環。根據製造商之規範分析PCR數據(Applied Biosystems關於ABI Prism 7700序列偵測系統之使用者佈告#2)。 Real-time PCR : Use Assays-on-Demand ® gene expression products to perform PCR on cDNA samples: use the 7000 sequence detection system (Applied Biosystems, Foster City, Calif.) With ABI Prism 7000 SDS software, according to the manufacturer ’s instructions (Applied Biosystems, Foster City, Calif.), Will oxidize LDL receptor (Hs00234028); rennet (Hs00166915); endoplasmic reticulum protein (Hs003825 15); CXC ligand 3 (Hs00171061); GCP-2 (Hs00605742); IL -8 (Hs00174103); (. Applied Biosystems, Foster City, Calif) , and mixed with GAPDH cDNA master mix and TaqMan ® Universal PCR. The thermal cycle conditions were to start 50 ° C for 2min and 95 ° C for 10min, followed by 40 cycles of 95 ° C for 15sec and 60 ° C for 1min. Analyze the PCR data according to the manufacturer's specifications (Applied Biosystems user notice # 2 on the ABI Prism 7700 sequence detection system).
傳統PCR:使用ABI PRISM 7700(Perkin Elmer Applied Biosystems,Boston,Mass.,USA)執行傳統PCR以確認即時PCR之結果。使用2微升cDNA溶液、1xAmpliTaq Gold通用混合物PCR反應緩衝液(Applied Biosystems,Foster City,Calif.)及在94℃下初始變性歷時5分鐘來執行PCR。擴增係針對各引子組最佳化。針 對IL-8、CXC配體3、及內質網蛋白(94℃歷時15秒、55℃歷時15秒及72℃歷時30秒持續30個循環);針對凝乳酶(94℃歷時15秒、53℃歷時15秒及72℃歷時30秒持續38個循環);針對氧化LDL受體與GAPDH(94℃歷時15秒、55℃歷時15秒及72℃歷時30秒持續33個循環)。擴增所使用之引子列舉於表11-1中。最終PCR反應中之引子濃度為1微莫耳,除了GAPDH為0.5微莫耳。GAPDH引子與即時PCR相同,除了未將製造商之TaqMan®探針添加至最終PCR反應。將樣本於2%(w/v)瓊脂糖凝膠上運行(run)且用溴化乙錠(Sigma,St.Louis,Mo.)染色。使用667 Universal Twinpack膜(VWR International,South Plainfield,N.J.),使用焦距Polaroid照相機(VWR International,South Plainfield,N.J.)擷取影像。 Traditional PCR : ABI PRISM 7700 (Perkin Elmer Applied Biosystems, Boston, Mass., USA) was used to perform traditional PCR to confirm the results of real-time PCR. PCR was performed using 2 microliters of cDNA solution, 1 x AmpliTaq Gold Universal Mix PCR reaction buffer (Applied Biosystems, Foster City, Calif.) And initial denaturation at 94 ° C for 5 minutes. The amplification line is optimized for each primer set. For IL-8, CXC ligand 3, and endoplasmic reticulum protein (15 seconds at 94 ° C, 15 seconds at 55 ° C, and 30 seconds at 72 ° C for 30 cycles); for rennet (15 seconds at 94 ° C, 53 ° C for 15 seconds and 72 ° C for 30 seconds for 38 cycles); for oxidized LDL receptor and GAPDH (94 ° C for 15 seconds, 55 ° C for 15 seconds and 72 ° C for 30 seconds for 33 cycles). The primers used for amplification are listed in Table 11-1. The primer concentration in the final PCR reaction was 1 micromolar, except for GAPDH which was 0.5 micromolar. The GAPDH primer is the same as real-time PCR, except that the manufacturer's TaqMan ® probe is not added to the final PCR reaction. The samples were run on a 2% (w / v) agarose gel and stained with ethidium bromide (Sigma, St. Louis, Mo.). Images were captured using a 667 Universal Twinpack film (VWR International, South Plainfield, NJ) and a focal length Polaroid camera (VWR International, South Plainfield, NJ).
免疫螢光:將PPDC在室溫下用冷4%(w/v)多聚甲醛(Sigma-Aldrich,St.Louis,Mo.)固定10分鐘。使用臍衍生細胞及胎盤衍生細胞各者在繼代0(P0)(分離後直接使用)的一個分離株,及臍衍生細胞及胎盤衍生細胞之繼代11(P 11)(兩個胎盤衍生細胞分離株、兩個臍衍生細胞分離株)及纖維母細胞(P 11)。使用針對以下表位的抗體執行免疫細胞化學:波形蛋白(1:500,Sigma,St.Louis,Mo.)、肌間線蛋白(1:150;Sigma--對抗兔而生成;或1:300;Chemicon,Temecula,Calif--對抗小鼠而生成)、α-平滑肌肌動蛋白(SMA;1:400;Sigma),細胞角蛋白18(CK18;1:400;Sigma),馮威里氏因子(vWF;1:200;Sigma)與CD34(人類CD34 Class III;1:100;DAKOCytomation,Carpinteria,Calif)。此外,於繼代11產後細胞上測試以下標記:抗人類GRO α--PE(1:100;Becton Dickinson,Franklin Lakes,N.J.)、抗人類GCP-2(1:100;Santa Cruz Biotech,Santa Cruz,Calif)、抗人類氧化LDL受體1(ox-LDL R1;1:100;Santa Cruz Biotech)與抗人類NOGA-A(1:100;Santa Cruz,Biotech)。 Immunofluorescence: PPDC was fixed with cold 4% (w / v) paraformaldehyde (Sigma-Aldrich, St. Louis, Mo.) at room temperature for 10 minutes. One isolate using the umbilical-derived cells and placenta-derived cells at passage 0 (P0) (used directly after isolation), and the passage 11 (P 11) (two placenta-derived cells) Isolates, two umbilical-derived cell isolates) and fibroblasts (P 11). Immunocytochemistry was performed using antibodies against the following epitopes: vimentin (1: 500, Sigma, St. Louis, Mo.), intermyosin (1: 150; Sigma--generated against rabbits; or 1: 300 ; Chemicon, Temecula, Calif-produced against mice), α-smooth muscle actin (SMA; 1: 400; Sigma), cytokeratin 18 (CK18; 1: 400; Sigma), von Willie ’s factor (vWF ; 1: 200; Sigma) and CD34 (human CD34 Class III; 1: 100; DAKOCytomation, Carpinteria, Calif). In addition, the following markers were tested on 11 post-natal cells: anti-human GRO α-PE (1: 100; Becton Dickinson, Franklin Lakes, NJ), anti-human GCP-2 (1: 100; Santa Cruz Biotech, Santa Cruz , Calif), anti-human oxidized LDL receptor 1 (ox-LDL R1; 1: 100; Santa Cruz Biotech) and anti-human NOGA-A (1: 100; Santa Cruz, Biotech).
用磷酸鹽緩衝液(PBS)清洗培養物然後將其暴露於含PBS、4%(v/v)山羊血清(Chemic on,Temecula,Calif)與0.3%(v/v)Triton(Triton X-100;Sigma,St.Louis,Mo.)的蛋白質阻斷液中30分鐘以獲取細胞內抗原。當感興趣表位係位於細胞表面(CD34、ox-LDL R1)上時,省略該程序的所有步驟中之Triton X-100以避免表位流失。再者,在一級抗體係對抗山羊(GCP-2、ox-LDL R1、NOGO-A)而生成的情況中,全程會使用3%(v/v)驢血清來取代山羊血清。接 著在室溫下將一級抗體(稀釋於阻斷液中)施用於培養物歷時1小時。將一級抗體溶液移除,用PBS清洗培養物,然後施用二級抗體溶液(在室溫下1小時),該二級抗體溶液含有阻斷劑以及山羊抗小鼠IgG--Texas Red(1:250;Molecular Probes,Eugene,Oreg.)及/或山羊抗兔IgG-Alexa 488(1:250;Molecular Probes)或驢抗山羊IgG--FITC(1:150,Santa Cruz Biotech)。隨後清洗培養物,然後施用10微莫耳DAPI(Molecular Probes)10分鐘以可視化細胞核。 The culture was washed with phosphate buffered saline (PBS) and then exposed to PBS, 4% (v / v) goat serum (Chemic on, Temecula, Calif) and 0.3% (v / v) Triton (Triton X-100 ; Sigma, St. Louis, Mo.) protein blocking solution for 30 minutes to obtain intracellular antigens. When the epitope of interest is located on the cell surface (CD34, ox-LDL R1), Triton X-100 in all steps of the procedure is omitted to avoid epitope loss. Furthermore, in the case where the primary anti-system is generated against goats (GCP-2, ox-LDL R1, NOGO-A), 3% (v / v) donkey serum will be used instead of goat serum throughout. Next, primary antibody (diluted in blocking solution) was applied to the culture at room temperature for 1 hour. The primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking agent and goat anti-mouse IgG--Texas Red (1: 250; Molecular Probes, Eugene, Oreg.) And / or goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes) or donkey anti-goat IgG-FITC (1: 150, Santa Cruz Biotech). The culture was subsequently washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.
在免疫染色後,在Olympus®倒立落射螢光顯微鏡(Olympus,Melville,N.Y.)上使用適當螢光濾光片來可視化螢光。在所有情況下,陽性染色代表高於對照組染色的螢光訊號,其中遵照上文所概述的全部程序,除了施加一級抗體溶液。使用數位彩色攝影機與ImagePro®軟體(Media Cybernetics,Carlsbad,Calif)來擷取代表影像。針對三重染色的樣本,一次僅使用一個發射濾光片來拍攝各影像。接著使用Adobe Photoshop®軟體(Adobe,San Jose,Calif)來製備分層合成影像(Layered montage)。 After immunostaining, use an appropriate fluorescent filter on an Olympus® inverted epifluorescence microscope (Olympus, Melville, N.Y.) to visualize the fluorescence. In all cases, positive staining represented a fluorescent signal higher than that of the control stain, in which all procedures outlined above were followed, except for the application of primary antibody solution. Use a digital color camera and ImagePro® software (Media Cybernetics, Carlsbad, Calif) to capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop® software (Adobe, San Jose, Calif) to prepare layered composite images (Layered montage).
製備細胞以進行FACS分析:將培養瓶中之貼附細胞於磷酸鹽緩衝液(PBS)(Gibco,Carlsbad,Calif)中清洗且用胰蛋白酶/EDTA(Gibco,Carlsbad,Calif)脫附。將細胞收集、離心,且以每毫升1×10 7的細胞濃度再懸浮於3%(v/v)FBS於PBS中。將一百微升等分遞送至錐形管中。細胞內抗原經染色之細胞係用Perm/Wash緩衝液(BD Pharmingen,San Diego,Calif)穿透。將抗體按製造商規範添加至等分中且將細胞在4℃下於黑暗中培養30分鐘。培養之後, 將細胞用PBS清洗並離心以移除過量抗體。將需要二級抗體之細胞再懸浮於100微升3% FBS中。將二級抗體按製造商規範添加且將細胞在4℃下於黑暗中培養30分鐘。培養之後,將細胞用PBS清洗並離心以移除過量二級抗體。將經清洗之細胞再懸浮於0.5毫升PBS中且藉由流動式細胞測量術分析。使用以下抗體:氧化LDL受體1(sc-5813;Santa Cruz,Biotech)、GROa(555042;BD Pharmingen,Bedford,Mass.)、小鼠IgG1κ(P-4685及M-5284;Sigma)、驢抗山羊IgG(sc-3743;Santa Cruz,Biotech.)。流動式細胞測量術分析係用FACScaliburTM(Becton Dickinson San Jose,Calif.)來執行。 Cells were prepared for FACS analysis: adherent cells in culture flasks were washed in phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif) and desorbed with trypsin / EDTA (Gibco, Carlsbad, Calif). The cells were collected, centrifuged, and resuspended in 3% (v / v) FBS in PBS at a cell concentration of 1 × 10 7 per ml. One hundred microliter aliquots were delivered into conical tubes. The stained cell lines with intracellular antigens were penetrated with Perm / Wash buffer (BD Pharmingen, San Diego, Calif). The antibody was added to aliquots according to the manufacturer's specifications and the cells were incubated at 4 ° C in the dark for 30 minutes. After incubation, the cells were washed with PBS and centrifuged to remove excess antibody. Resuspend cells that require secondary antibody in 100 μl of 3% FBS. Secondary antibodies were added according to the manufacturer's specifications and the cells were incubated in the dark at 4 ° C for 30 minutes. After incubation, the cells were washed with PBS and centrifuged to remove excess secondary antibody. The washed cells were resuspended in 0.5 ml PBS and analyzed by flow cytometry. The following antibodies were used: oxidized LDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa (555042; BD Pharmingen, Bedford, Mass.), Mouse IgG1κ (P-4685 and M-5284; Sigma), donkey anti Goat IgG (sc-3743; Santa Cruz, Biotech.). Flow cytometry analysis was performed with FACScalibur ™ (Becton Dickinson San Jose, Calif.).
在來自衍生自人類胎盤、成體及新生兒纖維母細胞與間葉幹細胞(MSC)之細胞的cDNA上所執行之針對選定「簽名」基因之即時PCR之結果指出,氧化LDL受體及凝乳酶兩者在胎盤衍生細胞中相較於其他細胞以較高水準表現。獲自即時PCR之數據係藉由AACT方法分析且以對數標度之形式表達。臍衍生細胞中內質網蛋白與氧化LDL受體表現水準相較於其他細胞為較高的。在產後衍生細胞與對照組之間未發現CXC配體3及GCP-2之表現水準有顯著差異。即時PCR之結果係藉由傳統PCR確認。PCR產物之定序進一步驗證這些觀察。使用上表19-1中所列舉之傳統PCR CXC配體3引子,發現產後衍生細胞與對照組之間CXC配體3之表現水準沒有顯著差異。 The results of real-time PCR for selected "signature" genes performed on cDNA from cells derived from human placenta, adult and neonatal fibroblasts and mesenchymal stem cells (MSC) indicate that the oxidized LDL receptor and curd Both enzymes perform at a higher level in placenta-derived cells than other cells. The data obtained from real-time PCR was analyzed by the AACT method and expressed on a log scale. The expression levels of endoplasmic reticulum protein and oxidized LDL receptor in umbilical cord-derived cells are higher than those in other cells. No significant differences were found in the performance levels of CXC ligand 3 and GCP-2 between the postpartum-derived cells and the control group. The results of real-time PCR are confirmed by conventional PCR. The sequencing of PCR products further validates these observations. Using the conventional PCR CXC ligand 3 primers listed in Table 19-1 above, it was found that there was no significant difference in the performance level of CXC ligand 3 between postpartum-derived cells and the control group.
產後細胞介素IL-8之生產在經生長培養基培養及經血清饑餓培養之產後衍生細胞兩者中均提高。所有即時PCR數據皆用傳統PCR及藉由定序PCR產物來驗證。 The production of postpartum cytokine IL-8 is increased in both the postpartum-derived cells cultured in growth medium and serum-starved. All real-time PCR data is verified using conventional PCR and by sequencing PCR products.
當檢測生長於無血清培養基中之細胞的上清液中IL-8之存在時,最高量在衍生自臍細胞及胎盤細胞之一些分離株之培養基中偵測出(表19-2)。在衍生自人類皮膚纖維母細胞之培養基中未偵測出IL-8。 When detecting the presence of IL-8 in the supernatant of cells grown in serum-free medium, the highest amount was detected in the culture medium of some isolates derived from umbilical cells and placental cells (Table 19-2). IL-8 was not detected in the culture medium derived from human skin fibroblasts.
亦藉由FACS分析檢測胎盤衍生細胞的氧化LDL受體、GCP-2及GROα之生產。經測試細胞為對GCP-2陽性。此方法未偵測到氧化LDL受體及GRO。 The production of oxidized LDL receptor, GCP-2 and GROα in placental-derived cells was also detected by FACS analysis. The tested cells were positive for GCP-2. This method did not detect oxidized LDL receptor and GRO.
亦藉由免疫細胞化學分析測試胎盤衍生細胞之選定蛋白質之生產。在分離後(繼代0),立即將衍生自人類胎盤之細胞用4%多聚甲醛固定且暴露於針對六種蛋白質之抗體:馮威里氏因 子、CD34、細胞角蛋白18、肌間線蛋白、α-平滑肌肌動蛋白、及波形蛋白。經染色細胞呈現α-平滑肌肌動蛋白及波形蛋白陽性。此模式直到繼代11仍保留。在繼代0時僅一些細胞(<5%)為細胞角蛋白18染色陽性。 The production of selected proteins of placenta-derived cells is also tested by immunocytochemical analysis. Immediately after isolation (subsequence 0), cells derived from human placenta were fixed with 4% paraformaldehyde and exposed to antibodies against six proteins: von Willie's factor, CD34, cytokeratin 18, intermycin, α-smooth muscle actin and vimentin. The stained cells were positive for α-smooth muscle actin and vimentin. This mode remains until generation 11. At passage 0, only some cells (<5%) were positive for cytokeratin 18 staining.
衍生自人類臍帶之細胞在繼代0時藉由免疫細胞化學分析探查到選定蛋白質之生產。在分離後(繼代0),立即將細胞用4%多聚甲醛固定且暴露於針對六種蛋白質之抗體:馮威里氏因子、CD34、細胞角蛋白18、肌間線蛋白、α-平滑肌肌動蛋白、及波形蛋白。臍衍生細胞為對α-平滑肌肌動蛋白及波形蛋白陽性,其中染色模式直到繼代11仍一致。 Cells derived from human umbilical cord were probed for the production of selected proteins by immunocytochemical analysis at 0 generations. Immediately after isolation (passage 0), the cells were fixed with 4% paraformaldehyde and exposed to antibodies against six proteins: von Willich's factor, CD34, cytokeratin 18, intermuscular protein, α-smooth muscle actin Protein, and vimentin. Umbilical-derived cells were positive for α-smooth muscle actin and vimentin, and the staining pattern remained consistent until passage 11.
總結:對於以下四種基因,已確立藉由微陣列及PCR(即時及傳統兩者)所量測之基因表現水準之間的一致性:氧化LDL受體1、凝乳酶、內質網蛋白、及IL-8。這些基因之表現在PPDC中的mRNA水準上受到差別調節,其中IL-8在蛋白質水準上亦受差別調節。藉由FACS分析,在衍生自胎盤之細胞中的蛋白質水準上未偵測出氧化LDL受體之存在。藉由FACS分析,GCP-2及CXC配體3於胎盤衍生細胞中在mRNA水準上之差別表現未經確認,但是在蛋白質水準上偵測出GCP-2。儘管此結果未由最初獲自微陣列實驗之數據反映出,但是此可能由於方法之靈敏度不同所致。 Summary : For the following four genes, the consistency between the gene expression levels measured by microarray and PCR (both real-time and traditional) has been established: oxidized LDL receptor 1, rennet, endoplasmic reticulum protein , And IL-8. The expression of these genes is differentially regulated at the mRNA level in PPDC, and IL-8 is also differentially regulated at the protein level. By FACS analysis, the presence of oxidized LDL receptors was not detected at the protein level in cells derived from the placenta. By FACS analysis, the differential performance of GCP-2 and CXC ligand 3 in placenta-derived cells at the mRNA level was not confirmed, but GCP-2 was detected at the protein level. Although this result is not reflected in the data originally obtained from the microarray experiment, it may be due to the different sensitivity of the method.
在分離後(繼代0),立即染色衍生自人類胎盤之細胞呈現α-平滑肌肌動蛋白及波形蛋白陽性。此模式亦在繼代11的細胞中觀察到。波形蛋白及α-平滑肌肌動蛋白表現可在繼代(在生長 培養基中及在這些程序中所利用之條件下)細胞中保留。在繼代0之衍生自人類臍帶之細胞係經探查α-平滑肌肌動蛋白及波形蛋白之表現,且兩者皆為陽性。此染色模式直到繼代11仍保留。 Immediately after isolation (Sub 0), cells derived from human placenta were stained positive for α-smooth muscle actin and vimentin. This pattern was also observed in cells of passage 11. Vimentin and α-smooth muscle actin expression can be retained in the subcultured cells (in the growth medium and under the conditions utilized in these procedures). The cell lines derived from human umbilical cord at passage 0 were tested for alpha-smooth muscle actin and vimentin, and both were positive. This staining pattern remains until generation 11.
體外評估產後衍生細胞(PPDC)之免疫特徵以預測這些細胞在體內移植後可能引發之免疫反應(若有的話)。藉由流動式細胞測量術檢定PPDC中HLA-DR、HLA-DP、HLA-DQ、CD80、CD86、及B7-H2之存在。這些蛋白質係由抗原呈現細胞(APe)表現且為純真(naïve)CD4+ T細胞之直接刺激所需(Abbas及Lichtman,CELLULAR AND MOLECULAR IMMUNOLOGY,第5版(2003)Saunders,Philadelphia,第171頁)。亦藉由流動式細胞測量術分析細胞系中HLA-G(Abbas及Lichtman,2003,見前文)、CD 178(Coumans等人,(1999)Journal of Immunological Methods 224,185-196)、及PD-L2(Abbas及Lichtman,2003,見前文;Brown等人(2003)The Journal of Immunology,170:1257-1266)之表現。認為存在於胎盤組織中的細胞所表現之這些蛋白質媒介子宮內胎盤組織之免疫豁免狀態。為了預測胎盤與臍衍生細胞系於體內引發免疫反應之程度,於單向混合淋巴球反應(MLR)中測試細胞系。 The immune characteristics of postpartum-derived cells (PPDC) are evaluated in vitro to predict the immune response (if any) that these cells may elicit after transplantation in vivo. The presence of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2 in PPDC was verified by flow cytometry. These proteins are expressed by antigen presenting cells (APe) and are required for direct stimulation of naïve CD4 + T cells (Abbas and Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th edition (2003) Saunders, Philadelphia, p. 171). Cell lines were also analyzed by flow cytometry for HLA-G (Abbas and Lichtman, 2003, see above), CD 178 (Coumans et al., (1999) Journal of Immunological Methods 224, 185-196), and PD-L2 ( Abbas and Lichtman, 2003, see above; Brown et al. (2003) The Journal of Immunology, 170: 1257-1266). It is believed that these proteins mediated by the cells present in the placental tissues mediate the immune exemption status of the intrauterine placental tissues. To predict the extent to which the placenta and umbilical-derived cell lines elicit an immune response in vivo, the cell lines were tested in a one-way mixed lymphocyte reaction (MLR).
細胞培養:於用2%明膠(Sigma,St.Louis,Mo.)塗布之T75瓶(Corning Inc.,Corning,N.Y.)中之含有青黴素/鏈黴素的生長培養基中培養細胞至長滿。 Cell culture: Cells were grown to fullness in growth medium containing penicillin / streptomycin in T75 flasks (Corning Inc., Corning, N.Y.) coated with 2% gelatin (Sigma, St. Louis, Mo.).
抗體染色:將細胞於磷酸鹽緩衝液(PBS)(Gibco,Carlsbad,Calif.)中清洗且用胰蛋白酶/EDTA(Gibco,Carlsbad,Mo.)脫附。將細胞收集、離心,且以每毫升1×107的細胞濃度再懸浮於3%(v/v)FBS於PBS中。按製造商規範將抗體(表20-1)添加至一百微升的細胞懸浮液中,且在4℃下於黑暗中培養30分鐘。培養之後,將細胞用PBS清洗並離心以移除未結合之抗體。將細胞再懸浮於五百微升PBS中且使用FACSCaliburTM儀器(Becton Dickinson,San Jose,Calif.)藉由流動式細胞測量術分析。 Antibody staining: cells were washed in phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif.) And desorbed with trypsin / EDTA (Gibco, Carlsbad, Mo.). The cells were collected, centrifuged, and resuspended in 3% (v / v) FBS in PBS at a cell concentration of 1 × 10 7 per ml. The antibody (Table 20-1) was added to one hundred microliters of cell suspension according to the manufacturer's specifications, and incubated at 4 ° C in the dark for 30 minutes. After incubation, the cells were washed with PBS and centrifuged to remove unbound antibody. The cells were resuspended in five hundred microliters of PBS and analyzed by flow cytometry using a FACSCalibur ™ instrument (Becton Dickinson, San Jose, Calif.).
混合淋巴球反應:將標記為細胞系A之繼代10臍衍生細胞與標記為細胞系B之繼代11胎盤衍生細胞的凍存小瓶於乾冰上送至CTBR(Senneville,Quebec)以使用CTBR SOP No.CAC-031 進行混合淋巴球反應。自多個男性及女性自願捐贈者收集周邊血液單核細胞(PBMC)。將刺激者(捐贈者)同種異體PBMC、自體PBMC、及產後細胞系用絲裂黴素C處理。將自體細胞及經絲裂黴素C處理之刺激者細胞添加至反應者(接受者)PBMC,並將其培養4天。在培養之後,將[3H]-胸苷添加至各樣本中且培養18小時。在收集細胞之後,萃取放射性標記DNA,且使用閃爍計數器量測[3H]-胸苷併入。 Mixed lymphocyte reaction: Freeze vials of 10 umbilical-derived cells labeled as cell line A and 11 placental-derived cells labeled as cell line B to CTBR (Senneville, Quebec) on dry ice to use CTBR SOP No.CAC-031 Perform mixed lymphocyte reaction. Peripheral blood mononuclear cells (PBMC) were collected from multiple male and female voluntary donors. The stimulator (donor) allogeneic PBMC, autologous PBMC, and postpartum cell lines were treated with mitomycin C. Autologous cells and stimulator cells treated with mitomycin C were added to the responder (recipient) PBMC and cultured for 4 days. After incubation, [ 3 H] -thymidine was added to each sample and incubated for 18 hours. After collecting the cells, the radiolabeled DNA was extracted, and [ 3 H] -thymidine incorporation was measured using a scintillation counter.
同種異體捐贈者刺激指數(SIAD)係經計算為:接收者之平均增生加上經絲裂黴素C處理之同種異體捐贈者之平均增生除以接收者之基線增生。PPDC刺激指數係經計算為:接收者之平均增生加上經絲裂黴素C處理之產後細胞系之平均增生除以接收者之基線增生。 The allogeneic donor stimulation index (SIAD) is calculated as the average hyperplasia of the recipient plus the average hyperplasia of the allogeneic donor treated with mitomycin C divided by the baseline hyperplasia of the recipient. The PPDC stimulation index is calculated as the average proliferation of the recipient plus the average proliferation of the postpartum cell line treated with mitomycin C divided by the baseline proliferation of the recipient.
混合淋巴球反應--胎盤衍生細胞:七位人類自願血液捐贈者經篩選以識別出在與其他六位血液捐贈者進行混合淋巴球反應時會展現穩固增生反應的單一同種異體捐贈者。此捐贈者係經選擇為同種異體陽性對照組捐贈者。剩餘六個血液捐贈者係選擇為接受者。將同種異體陽性對照組捐贈者及胎盤衍生細胞系用絲裂黴素C處理且與六個個別同種異體接收者培養於混合淋巴球反應中。反應係使用兩個細胞培養盤重複三次執行,每盤具有三個接收者(表 20-2)。平均刺激指數之範圍為1.3(盤2)至3(盤1),且同種異體捐贈者陽性對照組之範圍為46.25(盤2)至279(盤1)(表20-3)。 Mixed Lymphocyte Response-Placenta-derived cells: Seven human voluntary blood donors were screened to identify a single allogeneic donor who would exhibit a solid hyperplastic response when mixed lymphocyte response was performed with six other blood donors This donor was selected as the allogeneic positive control group donor. The remaining six blood donors were selected as recipients. Allogeneic positive control donors and placenta-derived cell lines were treated with mitomycin C and cultured in a mixed lymphocyte reaction with six individual allogeneic recipients. The reaction system was repeated three times using two cell culture plates, each with three recipients (Table 20-2). The average stimulation index ranged from 1.3 (disc 2) to 3 (disc 1), and the positive control group of allogeneic donors ranged from 46.25 (disc 2) to 279 (disc 1) (Table 20-3).
混合淋巴球反應--臍衍生細胞:六位人類自願血液捐贈者經篩選以識別出在與其他五位血液捐贈者進行混合淋巴球反應時會展現穩固增生反應的單一同種異體捐贈者。此捐贈者係經選擇為同種異體陽性對照組捐贈者。剩餘五個血液捐贈者係選擇為接受者。將同種異體陽性對照組捐贈者及胎盤細胞系用絲裂黴素C處理且與五個個別同種異體接收者培養於混合淋巴球反應中。反應係使用兩個細胞培養盤重複三次執行,每盤具有三個接收者(表12-4)。平均刺激指數之範圍為6.5(盤1)至9(盤2),且同種異體捐贈者陽性對照組之範圍為42.75(盤1)至70(盤2)(表12-5)。 Mixed Lymphocyte Response-Umbilical Derived Cells: Six human voluntary blood donors were screened to identify a single allogeneic donor who exhibited a solid hyperplastic response when mixed lymphocyte responses were performed with five other blood donors. This donor was selected as the allogeneic positive control group donor. The remaining five blood donors were selected as recipients. Allogeneic positive control donors and placental cell lines were treated with mitomycin C and cultured in a mixed lymphocyte reaction with five individual allogeneic recipients. The reaction system was repeated three times using two cell culture plates, each with three recipients (Table 12-4). The average stimulation index ranged from 6.5 (disc 1) to 9 (disc 2), and the positive control group of allogeneic donors ranged from 42.75 (disc 1) to 70 (disc 2) (Table 12-5).
表20-5.臍帶衍生細胞及同種異體捐贈者在與五個個別同種異體接
抗原呈現細胞標記--胎盤衍生細胞:藉由流動式細胞測量術所分析之胎盤衍生細胞的直方圖顯示HLA-DR、HLA-DP、HLA-DQ、CD80、CD86、及B7-H2之陰性表現,如藉由與IgG對照組一致的螢光值所說明,其指示胎盤細胞系缺乏直接刺激CD4+ T細胞所需之細胞表面分子。 Antigen-presenting cell markers-placenta-derived cells: Histograms of placenta-derived cells analyzed by flow cytometry showed negative performance of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2 As indicated by the fluorescence values consistent with the IgG control group, it indicates that the placental cell line lacks the cell surface molecules required to directly stimulate CD4 + T cells.
免疫調節標記--胎盤衍生細胞:藉由流動式細胞測量術所分析之胎盤衍生細胞的直方圖顯示PD-L2之陽性表現,如藉由相對於IgG對照組螢光值增加所說明,以及CD178及HLA-G之陰性表現,如藉由與IgG對照組一致的螢光值所說明。 Immunomodulatory markers-placenta-derived cells: Histograms of placenta-derived cells analyzed by flow cytometry showed a positive manifestation of PD-L2, as illustrated by the increase in fluorescence relative to the IgG control group, and CD178 And the negative performance of HLA-G, as illustrated by the fluorescence value consistent with the IgG control group.
抗原呈現細胞標記--臍衍生細胞:藉由流動式細胞測量術所分析之臍衍生細胞的直方圖顯示HLA-DR、HLA-DP、HLA-DQ、CD80、CD86、及B7-H2之陰性表現,如藉由與IgG對照組一致的螢光值所說明,其指示臍細胞系缺乏直接刺激CD4+ T細胞所需之細胞表面分子。 Antigen-presenting cell markers-umbilical derived cells: Histogram of umbilical derived cells analyzed by flow cytometry showed negative performance of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2 As indicated by the fluorescence values consistent with the IgG control group, it indicates that the umbilical cell line lacks the cell surface molecules required to directly stimulate CD4 + T cells.
免疫調節細胞標記--臍衍生細胞:藉由流動式細胞測量術所分析之臍衍生細胞的直方圖顯示PD-L2之陽性表現,如藉由相對於IgG對照組螢光值增加所說明,以及CD178及HLA-G之陰性表現,如藉由與IgG對照組一致的螢光值所說明。 Immune Regulatory Cell Marker-Umbilical-derived cells: The histogram of umbilical-derived cells analyzed by flow cytometry shows a positive performance of PD-L2, as illustrated by the increase in fluorescence value relative to the IgG control group, and The negative performance of CD178 and HLA-G was explained by the fluorescence values consistent with the IgG control group.
總結:在用胎盤衍生細胞系進行之混合淋巴球反應中,平均刺激指數之範圍為1.3至3,且同種異體陽性對照組之平均刺激指數之範圍為46.25至279。在用臍衍生細胞系進行之混合淋巴球反應中,平均刺激指數之範圍為6.5至9,且同種異體陽性對照組之平均刺激指數之範圍為42.75至70。胎盤及臍衍生細胞系對於刺激蛋白質HLA-DR、HLA-DP、HLA-DQ、CD80、CD86、及B7-H2之表現為陰性,如藉由流動式細胞測量術所量測。胎盤及臍衍生細胞系對於免疫調節蛋白質HLA-G及CD178之表現為陰性且對於PD-L2之表現為陽性,如藉由流動式細胞測量術所量測。同種異體捐贈者PBMC含有表現HLA-DR、HLA-DQ、CD8、CD86、及B 7-H2之抗原呈現細胞,從而允許純真CD4+ T細胞之刺激。胎盤及臍衍生細胞上不存在直接刺激純真CD4+ T細胞所需之抗原呈現細胞表面分子及存在免疫調節蛋白質PD-L2,可說明於MLR中相較於同種異體對照組,這些細胞所展現之刺激指數較低。 Summary: In the mixed lymphocyte reaction with placenta-derived cell lines, the average stimulation index ranged from 1.3 to 3, and the allogeneic positive control group had an average stimulation index range from 46.25 to 279. In the mixed lymphocyte reaction with the umbilical-derived cell line, the average stimulation index ranged from 6.5 to 9, and the allogeneic positive control group had an average stimulation index ranging from 42.75 to 70. The placenta and umbilical-derived cell lines are negative for the stimulation proteins HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2, as measured by flow cytometry. The placenta and umbilical derived cell lines were negative for the immunomodulatory proteins HLA-G and CD178 and positive for PD-L2, as measured by flow cytometry. Allogeneic donor PBMC contains antigen-presenting cells expressing HLA-DR, HLA-DQ, CD8, CD86, and B 7-H2, thereby allowing the stimulation of pure CD4 + T cells. There is no antigen present on the placenta and umbilical-derived cells that directly stimulates pure CD4 + T cells to present cell surface molecules and the presence of the immunomodulatory protein PD-L2. The index is low.
對於來自胎盤及臍衍生細胞之選定營養因子的分泌係經量測。所偵測之選定因子包括:(1)已知具有血管生成活性之因子,諸如肝細胞生長因子(HGF)(Rosen等人(1997)Ciba Found.Symp.212:215-26)、單核細胞趨化蛋白質1(MCP-1)(Salcedo等人(2000)Blood 96;34-40)、介白素-8(IL-8)(Li等人(2003)J. Immunol.170:3369-76)、角質細胞生長因子(KGF)、鹼性纖維母細胞生長因子(bFGF)、血管內皮生長因子(VEGF)(Hughes等人(2004)Ann.Thorac.Surg.77:812-8)、基質金屬蛋白酶1(TIMP1)、血管生成素2(ANG2)、血小板衍生生長因子(PDGF-bb)、血小板生成素(TPO)、肝素結合表皮生長因子(HB-EGF)、基質衍生因子1α(SDF-1α);(2)已知具有神經營養/神經保護活性之因子,諸如腦衍生神經營養因子(BDNF)(Cheng等人(2003)Dev.Biol.258;319-33)、介白素-6(IL-6)、顆粒性細胞趨化蛋白質-2(GCP-2)、轉形生長因子β2(TGFβ2);及(3)已知具有趨化激素活性之因子,諸如巨噬細胞發炎蛋白質1α(MIP1a)、巨噬細胞發炎蛋白質1β(MIP1b)、單核細胞趨化因子-1(MCP-1)、Rantes(調控活化、正常T細胞表現及分泌)、I309、胸腺與活化調控趨化激素(TARe)、伊紅趨素(Eotaxin)、巨噬細胞衍生趨化激素(MDC)、IL-8。 The secretion of selected nutrient factors from placenta and umbilical derived cells was measured. Selected factors detected include: (1) Factors known to have angiogenic activity, such as hepatocyte growth factor (HGF) (Rosen et al. (1997) Ciba Found. Symp. 212: 215-26), monocytes Chemotactic protein 1 (MCP-1) (Salcedo et al. (2000) Blood 96; 34-40), interleukin-8 (IL-8) (Li et al. (2003) J. Immunol. 170: 3369-76 ), Keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) (Hughes et al. (2004) Ann. Thorac. Surg. 77: 812-8), matrix metal Protease 1 (TIMP1), angiopoietin 2 (ANG2), platelet-derived growth factor (PDGF-bb), thrombopoietin (TPO), heparin-binding epidermal growth factor (HB-EGF), matrix-derived factor 1α (SDF-1α ); (2) Factors known to have neurotrophic / neuroprotective activity, such as brain-derived neurotrophic factor (BDNF) (Cheng et al. (2003) Dev. Biol. 258; 319-33), interleukin-6 ( IL-6), Granular Cell Chemotactic Protein-2 (GCP-2), Transforming Growth Factor β2 (TGFβ2); and (3) Factors known to have chemokine activity, such as macrophage inflamed protein 1α ( MIP1a), macrophage inflammation protein 1 β (MIP1b), monocyte chemokine-1 (MCP-1), Rantes (regulatory activation, normal T cell expression and secretion), I309, thymus and activation-regulated chemokine (TARe), erythropoietin ( Eotaxin), macrophage-derived chemokine (MDC), IL-8.
細胞培養:將來自胎盤及臍之PPDC以及衍生自人類新生兒包皮之人類纖維母細胞培養於經明膠塗布之T75培養瓶中的具有青黴素/鏈黴素之生長培養基中。將細胞在繼代11時凍存並儲存於液態氮中。在解凍細胞後,將生長培養基加至細胞,接著轉移至15毫升離心管中然後以150xg離心細胞5分鐘。捨棄上清液。將細胞沉澱物再懸浮於4毫升生長培養基中,然後計數細胞。使細胞以375,000個細胞/75cm2接種於含有15毫升生長培養基之培養瓶中 且培養24小時。將培養基更換為無血清培養基(DMEM-低葡萄糖(Gibco)、0.1%(w/v)牛血清白蛋白(Sigma)、青黴素/鏈黴素(Gibco))歷時8小時。在培養結束時以14,000xg離心5分鐘來收集無血清條件培養基,然後儲存在-20℃下。 Cell culture: PPDC from placenta and umbilical cord and human fibroblasts derived from human neonatal foreskin are cultured in growth medium with penicillin / streptomycin in gelatin-coated T75 flasks. The cells were frozen at 11 o'clock and stored in liquid nitrogen. After thawing the cells, the growth medium was added to the cells, then transferred to a 15 ml centrifuge tube and the cells were centrifuged at 150 x g for 5 minutes. Discard the supernatant. The cell pellet was resuspended in 4 ml of growth medium, and the cells were counted. The cells were seeded at 375,000 cells / 75 cm 2 in a culture flask containing 15 ml of growth medium and cultured for 24 hours. The medium was replaced with serum-free medium (DMEM-low glucose (Gibco), 0.1% (w / v) bovine serum albumin (Sigma), penicillin / streptomycin (Gibco)) for 8 hours. At the end of the culture, centrifuge at 14,000 x g for 5 minutes to collect serum-free conditioned medium, and then store at -20 ° C.
為了要評估各培養瓶中的細胞數目,用PBS清洗細胞然後使用2毫升胰蛋白酶/EDTA脫附。藉由加入8毫升生長培養基來抑制胰蛋白酶活性。將細胞以150xg離心5分鐘。將上清液移除然後將細胞再懸浮於1毫升的生長培養基中。使用血球計估計細胞數目。 To assess the number of cells in each flask, wash the cells with PBS and then use 2 ml of trypsin / EDTA for desorption. Trypsin activity was inhibited by adding 8 ml of growth medium. Centrifuge the cells at 150 x g for 5 minutes. The supernatant was removed and the cells were resuspended in 1 ml of growth medium. Estimate the cell number using a hemocytometer.
ELISA檢定:使細胞在37℃下生長於5%二氧化碳及大氣氧氣中。亦使胎盤衍生細胞(批次101503)生長於5%氧氣或β-巰基乙醇(BME)中。各細胞樣本所生產之MCP-1、IL-6、VEGF、SDF-1α、GCP-2、IL-8及TGF-β2之量係以ELISA檢定(R&D Systems,Minneapolis,Mn.)來量測。所有檢定皆依據製造商說明書來執行。 ELISA test: cells are grown in 5% carbon dioxide and atmospheric oxygen at 37 ° C. Placental-derived cells (batch 101503) were also grown in 5% oxygen or β-mercaptoethanol (BME). The amount of MCP-1, IL-6, VEGF, SDF-1α, GCP-2, IL-8 and TGF-β2 produced by each cell sample was measured by ELISA (R & D Systems, Minneapolis, Mn.). All verifications are performed according to the manufacturer's instructions.
SearchLight TM多工ELISA檢定:趨化激素(MIP1a、MIP1b、MCP-1、Rantes、1309、TARC、伊紅趨素(Eotaxin)、MDC、IL8)、BDNF、及血管生成因子(HGF、KGF、bFGF、VEGF、TIMP1、ANG2、PDGF-bb、TPO、HB-EGF)皆使用searchLightTM Proteome Arrays(Pierce Biotechnology Inc.)來測量。Proteome Arrays為多工三明治法ELISA,用於進行每孔二至16種蛋白質的定量量測。藉由將四至16種不同捕捉抗體(capture antibody)以2×2、3×3、或4×4圖案點樣至96孔盤的各孔中而生產陣列。在進行三明治法ELISA 程序後,將整個盤成像以捕捉盤上各孔內之各點樣處所產生的化學發光信號。在各點樣處所產生的信號量係正比於原始標準品或樣本中的目標蛋白質之量。 SearchLight TM multiplex ELISA test: chemokines (MIP1a, MIP1b, MCP-1, Rantes, 1309, TARC, Eotaxin, MDC, IL8), BDNF, and angiogenic factors (HGF, KGF, bFGF , VEGF, TIMP1, ANG2, PDGF-bb, TPO, HB-EGF) were measured using searchLight ™ Proteome Arrays (Pierce Biotechnology Inc.). Proteome Arrays is a multiplex sandwich ELISA for quantitative measurement of two to 16 proteins per well. Arrays were produced by spotting four to 16 different capture antibodies into each well of a 96-well plate in a 2 × 2, 3 × 3, or 4 × 4 pattern. After the sandwich ELISA procedure, the entire disk was imaged to capture the chemiluminescent signal generated at each spot in each well on the disk. The amount of signal generated at each spot is proportional to the amount of target protein in the original standard or sample.
ELISA檢定:MCP-1及IL-6係由胎盤衍生細胞及臍衍生細胞以及皮膚纖維母細胞分泌(表21-1)。SDF-1α係由培養於5% O2中之胎盤衍生細胞及纖維母細胞所分泌。GCP-2及IL-8係由臍衍生細胞及在BME或5% O2存在下培養之胎盤衍生細胞所分泌。GCP-2亦由人類纖維母細胞所分泌。TGF-β2未由ELISA檢定偵測出。 ELISA test: MCP-1 and IL-6 lines are secreted by placental-derived cells, umbilical-derived cells, and skin fibroblasts (Table 21-1). SDF-1α is secreted by placental-derived cells and fibroblasts cultured in 5% O 2 . GCP-2 and IL-8 are secreted by navel-derived cells and placental-derived cells cultured in the presence of BME or 5% O 2 . GCP-2 is also secreted by human fibroblasts. TGF-β2 was not detected by ELISA test.
SearchLight TM多工ELISA檢定:TIMP1、TPO、KGF、HGF、FGF、HBEGF、BDNF、MIP1b、MCP1、RANTES、I309、TARC、 MDC、及IL-8皆分泌自臍衍生細胞(表21-2及表21-3)。TIMP1、TPO、KGF、HGF、HBEGF、BDNF、MIP1a、MCP-1、RANTES、TARC、Eotaxin、與IL-8皆分泌自胎盤衍生細胞(表21-2及21-3)。未偵測到Ang2、VEGF或PDGF-bb。 SearchLight TM multiplex ELISA test: TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1, RANTES, I309, TARC, MDC, and IL-8 are all secreted from umbilical derived cells (Table 21-2 and Table 21-3). TIMP1, TPO, KGF, HGF, HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 are all secreted from placental-derived cells (Tables 21-2 and 21-3). No Ang2, VEGF or PDGF-bb were detected.
檢測胎盤與臍衍生細胞(統稱為產後衍生細胞或PPDC)分化成神經譜系細胞之能力。 The ability of placenta and umbilical derived cells (collectively called postpartum derived cells or PPDC) to differentiate into neural lineage cells was tested.
產後細胞之分離及擴增:將來自胎盤及臍組織之PPDC如實例14所述分離並擴增。 Isolation and expansion of postpartum cells: PPDCs from placenta and umbilical tissue were isolated and expanded as described in Example 14.
修改的伍德柏-布萊克(Woodbury-Black)規程(A):此檢定係改編自最初執行以測試骨髓基質細胞之神經誘導潛能(1)之檢定。將臍衍生細胞(022803)P4與胎盤衍生細胞(042203)P3解凍且以5,000個細胞/cm2培養擴增於生長培養基中直到達到次長滿(75%)。隨後將細胞進行胰蛋白酶消化且以每孔6,000個細胞接種於Titretek II玻片(VWR International,Bristol,Conn.)上。在對照組方面,間葉幹細胞(P3;1F2155;Cambrex,Walkersville,Md.)、造骨細胞(P5;CC2538;Cambrex)、脂肪衍生細胞(Artecel,美國專利第6,555,374 B1號)(P6;捐贈者2)與新生兒人類皮膚纖維母細胞(P6;CC2509;Cambrex)亦在相同條件下接種。 Modified Woodbury-Black procedure (A) : This test is adapted from the test that was originally performed to test the nerve-inducing potential of bone marrow stromal cells (1). Umbilical-derived cells (022803) P4 and placenta-derived cells (042203) P3 were thawed and culture-expanded at 5,000 cells / cm 2 in growth medium until reaching sub-fullness (75%). The cells were then trypsinized and seeded on Titretek II slides (VWR International, Bristol, Conn.) At 6,000 cells per well. In the control group, mesenchymal stem cells (P3; 1F2155; Cambrex, Walkersville, Md.), Osteoblasts (P5; CC2538; Cambrex), fat-derived cells (Artecel, US Patent No. 6,555,374 B1) (P6; donor 2) Inoculated under the same conditions as neonatal human skin fibroblasts (P6; CC2509; Cambrex).
最初將所有細胞於DMEM/F12培養基(Invitrogen,Carlsbad,Calif.)中擴增4天,該培養基含有15%(v/v)胎牛血清(FBS;Hyclone,Logan,Utah)、鹼性纖維母細胞生長因子(bFGF;20奈克/毫升;Peprotech,Rocky Hill,N.J.)、表皮生長因子(EGF;20奈克/毫升;Peprotech)與青黴素/鏈黴素(Invitrogen)。四天後,將細胞於磷酸鹽緩衝液(PBS;Invitrogen)中潤洗且隨後培養於DMEM/F12培養基+20%(v/v)FBS+青黴素/鏈黴素中24小時。24小時後,將細胞用PBS潤洗。隨後使細胞於包含DMEM/FI2(無血清)之誘導培養 基中培養1至6小時,該培養基含有200mM丁基化羥基甲氧苯、10μM氯化鉀、5毫克/毫升胰島素、10μM毛喉素(forskolin)、4μM丙戊酸、與2μM氫皮質酮(所有化學品皆來自Sigma,St.Louis,Mo.)。隨後將細胞固定於100%冰冷甲醇中且執行免疫細胞化學(參見以下方法)以評估人類巢蛋白表現。 Initially all cells were expanded for 4 days in DMEM / F12 medium (Invitrogen, Carlsbad, Calif.) Containing 15% (v / v) fetal bovine serum (FBS; Hyclone, Logan, Utah), basic fiber mother Cell growth factor (bFGF; 20 ng / ml; Peprotech, Rocky Hill, NJ), epidermal growth factor (EGF; 20 ng / ml; Peprotech) and penicillin / streptomycin (Invitrogen). After four days, the cells were rinsed in phosphate buffer (PBS; Invitrogen) and then cultured in DMEM / F12 medium + 20% (v / v) FBS + penicillin / streptomycin for 24 hours. After 24 hours, cells were rinsed with PBS. The cells were then cultured for 1 to 6 hours in an induction medium containing DMEM / FI2 (serum-free) containing 200 mM butylated hydroxymethoxybenzene, 10 μM potassium chloride, 5 mg / ml insulin, and 10 μM forskolin ( forskolin), 4 μM valproic acid, and 2 μM hydrocorticosterone (all chemicals are from Sigma, St. Louis, Mo.). Cells were then fixed in 100% ice-cold methanol and immunocytochemistry (see method below) was performed to evaluate human nestin performance.
修改的伍德柏-布萊克規程(B):將PPDC(臍(022803)P11;胎盤(042203)P11)及成體人類皮膚纖維母細胞(1F1853,P11)解凍,並以5,000個細胞/cm2培養擴增於生長培養基中直到達到次長滿(75%)。接著將細胞進行胰蛋白酶消化並以與(A)中類似的密度接種,但係接種至(1)24孔經組織培養物處理之盤(TCP,Falcon brand,VWR International)、(2)TCP孔+2%(w/v)明膠(在室溫下吸附1小時)、或(3)TCP孔+20μg/毫升吸附小鼠層黏蛋白(在37℃下吸附最少2小時;Invitrogen)。 Modified Woodbury-Black protocol (B) : PPDC (umbilical (022803) P11; placental (042203) P11) and adult human skin fibroblasts (1F1853, P11) are thawed and cultured at 5,000 cells / cm 2 Amplify in growth medium until reaching sub-fullness (75%). The cells were then trypsinized and seeded at a similar density as in (A), but inoculated to (1) 24-well tissue culture treated discs (TCP, Falcon brand, VWR International), (2) TCP wells + 2% (w / v) gelatin (adsorbed for 1 hour at room temperature), or (3) TCP well +20 μg / ml for adsorbed mouse laminin (adsorbed at 37 ° C for a minimum of 2 hours; Invitrogen).
和(A)中完全相同,細胞最初係經擴增且在前述時間框下轉換培養基。如前述在5天6小時固定一組培養物,但此次在室溫下使用冰冷的4%(w/v)多聚甲醛(Sigma)歷時10分鐘。在第二組培養物中,將培養基移除並轉換成神經前驅擴增培養基(NPE),其係由Neurobasal-A培養基(Invitrogen)所組成,該Neurobasal-A培養基含有B27(B27補充物;Invitrogen)、L-麩醯胺酸(4mM)、及青黴素/鏈黴素(Invitrogen)。將NPE培養基用視黃酸進一步補充(RA;1μM;Sigma)。4天後將此培養基移除,且在室溫下將培養物用冰冷的4% (w/v)多聚甲醛(Sigma)固定10分鐘,並針對巢蛋白、GFAP、及TuJ1蛋白質表現進行染色(參見表22-1)。 As in (A), the cells were initially expanded and the medium was switched in the aforementioned time frame. A group of cultures was fixed as described above at 5 days and 6 hours, but this time using ice-cold 4% (w / v) paraformaldehyde (Sigma) at room temperature for 10 minutes. In the second set of cultures, the medium was removed and converted to neural precursor expansion medium (NPE), which consisted of Neurobasal-A medium (Invitrogen), which contained B27 (B27 supplement; Invitrogen ), L-glutamic acid (4mM), and penicillin / streptomycin (Invitrogen). The NPE medium was further supplemented with retinoic acid (RA; 1 μM; Sigma). After 4 days, the medium was removed, and the culture was fixed with ice-cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and stained for nestin, GFAP, and TuJ1 protein expression (See Table 22-1).
兩階段分化規程:將PPDC(臍(042203)P11、胎盤(022803)P11)、成體人類皮膚纖維母細胞(P11;1F1853;Cambrex)解凍且以5,000個細胞/cm2培養擴增於生長培養基中直到達到次長滿(75%)。接著將細胞進行胰蛋白酶消化並以2,000個細胞/cm2接種,但係至接種有NPE培養基存在之24孔經層黏蛋白塗布之盤(BD Biosciences,Franklin Lakes,N.J.)上,該NPE培養基補充有bFGF(20奈克/毫升;Peprotech,Rocky Hill,N.J)及EGF(20奈克/毫升;Peprotech)[整個培養基組成物進一步稱為NPE+F+E]。在相同時間,亦將自海馬迴分離之成鼠神經前驅(P4;062603)接種至24孔經層黏蛋白塗布之盤(於NPE+F+E培養基中)。將所有培養物維持在此類條件下歷時6天之時期(在該時間期間餵養細胞一次),此時將培養基轉換成表14-2中所列舉之分化條件再歷時7天之時期。將培養 物在室溫下用冰冷的4%(w/v)多聚甲醛(Sigma)固定10分鐘,且針對大鼠巢蛋白、GF AP、及TuJ1蛋白質表現進行染色。 Two-stage differentiation protocol: PPDC (umbilical (042203) P11, placental (022803) P11), adult human skin fibroblasts (P11; 1F1853; Cambrex) are thawed and cultured at 5,000 cells / cm 2 in growth medium Medium until reaching the second overgrown (75%). The cells were then trypsinized and seeded at 2,000 cells / cm 2 , but tied to a 24-well laminin-coated disk (BD Biosciences, Franklin Lakes, NJ) inoculated with NPE medium supplemented with this NPE medium There are bFGF (20 ng / ml; Peprotech, Rocky Hill, NJ) and EGF (20 ng / ml; Peprotech) [the entire medium composition is further referred to as NPE + F + E]. At the same time, the adult rat neural precursor (P4; 062603) isolated from the hippocampus was also inoculated into a 24-well laminin-coated disk (in NPE + F + E medium). All cultures were maintained under such conditions for a period of 6 days (the cells were fed once during this period), at which time the medium was switched to the differentiation conditions listed in Table 14-2 for a period of another 7 days. Cultures were fixed with ice-cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and stained for rat nestin, GF AP, and TuJ1 protein performance.
多重生長因子規程:將臍衍生細胞(P11;(042203))解凍並以5,000個細胞/cm2培養擴增於生長培養基中直到達到次長滿(75%)。隨後將細胞進行胰蛋白酶消化且以2,000個細胞/cm2接種至有NPE+F(20奈克/毫升)+E(20奈克/毫升)存在之24孔經層黏蛋白塗布之盤(BD Biosciences)上。此外,一些細胞含有NPE+F+E+2% FBS或10% FBS。在四天「分化前(pre-differentiation)」條件之後,將所有培養基移除且將樣本轉換成NPE培養基,該培養 基補充有聲蝟(SHH;200奈克/毫升;Sigma,St.Louis,Mo.)、FGF8(100奈克/毫升;Peprotech)、BDNF(40奈克/毫升;Sigma)、GDNF(20奈克/毫升;Sigma)、及視黃酸(1μM;Sigma)。在培養基更換後七天,在室溫下將培養物用冰冷的4%(w/v)多聚甲醛(Sigma)固定10分鐘,且針對人類巢蛋白、GFAP、TuJ1、肌間線蛋白、及α-平滑肌肌動蛋白表現進行染色。 Multiple growth factor protocol: Umbilical derived cells (P11; (042203)) were thawed and cultured at 5,000 cells / cm 2 in growth medium until reaching the second fullness (75%). The cells were then trypsinized and seeded at 2,000 cells / cm 2 into a 24-well laminin-coated dish (BD) in the presence of NPE + F (20 ng / ml) + E (20 ng / ml) Biosciences). In addition, some cells contain NPE + F + E + 2% FBS or 10% FBS. After four days of "pre-differentiation" conditions, all media was removed and the samples were converted to NPE media supplemented with acoustic hedgehog (SHH; 200 ng / ml; Sigma, St. Louis, Mo. ), FGF8 (100 ng / ml; Peprotech), BDNF (40 ng / ml; Sigma), GDNF (20 ng / ml; Sigma), and retinoic acid (1 μM; Sigma). Seven days after the medium change, the culture was fixed with ice-cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and targeted to human nestin, GFAP, TuJ1, intermyosin, and alpha -Staining of smooth muscle actin performance.
神經前驅共培養規程:將成鼠海馬廻前驅(062603)以神經球或單一細胞(10,000個細胞/孔)接種至經層黏蛋白塗布之24孔盤(BD Biosciences)上之NPE+F(20奈克/毫升)+E(20奈克/毫升)中。 Neural precursor co-cultivation protocol: Inoculate adult rat hippocampus hippocampus precursor (062603) with neurospheres or single cells (10,000 cells / well) onto NPE + F (20 Nanograms / ml) + E (20 nanograms / ml).
分開地,將臍衍生細胞(042203)P11與胎盤衍生細胞(022803)P11解凍且以5,000個細胞/cm2培養擴增於NPE+F(20奈克/毫升)+E(20奈克/毫升)中歷時48小時之時期。隨後將細胞進行胰蛋白酶消化且以2,500個細胞/孔接種至神經前驅之現有培養物上。此時,將現有培養基交換成新鮮培養基。四天後,將培養物在室溫下用冰冷的4%(w/v)多聚甲醛(Sigma)固定10分鐘,並針對人類核蛋白(hNuc;Chemicon)進行染色(上文表14-1)以識別PPDC。 Separately, umbilical-derived cells (042203) P11 and placenta-derived cells (022803) P11 were thawed and cultured and expanded at 5,000 cells / cm 2 in NPE + F (20 ng / ml) + E (20 ng / ml) ) In the 48-hour period. The cells were then trypsinized and seeded at 2,500 cells / well onto existing neural precursor cultures. At this time, the existing medium is exchanged for fresh medium. Four days later, the culture was fixed with ice-cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature and stained for human nuclear protein (hNuc; Chemicon) (Table 14-1 above) ) To identify PPDC.
免疫細胞化學:使用表14-1中所列舉之抗體執行免疫細胞化學。用磷酸鹽緩衝液(PBS)清洗培養物然後將其暴露於含PBS、4%(v/v)山羊血清(Chemicon,Temecula,Calif)與0.3%(v/v)Triton(Triton X-100;Sigma)的蛋白質阻斷液中30分鐘以獲取細胞內抗原。接著在室溫下將一級抗體(稀釋於阻斷液中)施用於培養 物歷時1小時。接著,將一級抗體溶液移除,用PBS清洗培養物,然後施用二級抗體溶液(在室溫下1小時),該二級抗體溶液含有阻斷溶液以及山羊抗小鼠IgG--Texas Red(1:250;Molecular Probes,Eugene,OR)及山羊抗兔IgG-Alexa 488(1:250;Molecular Probes)。隨後清洗培養物,然後施用10微莫耳DAPI(Molecular Probes)10分鐘以可視化細胞核。 Immunocytochemistry: Immunocytochemistry was performed using the antibodies listed in Table 14-1. The culture was washed with phosphate buffered saline (PBS) and then exposed to PBS, 4% (v / v) goat serum (Chemicon, Temecula, Calif) and 0.3% (v / v) Triton (Triton X-100; Sigma) protein blocking solution for 30 minutes to obtain intracellular antigens. Next, primary antibody (diluted in blocking solution) was applied to the culture at room temperature for 1 hour. Next, the primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking solution and goat anti-mouse IgG--Texas Red ( 1: 250; Molecular Probes, Eugene, OR) and goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes). The culture was subsequently washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.
在免疫染色後,在Olympus倒立落射螢光顯微鏡(Olympus,Melville,N.Y.)上使用適當螢光濾光片來可視化螢光。在所有情況下,陽性染色代表高於對照組染色的螢光訊號,其中遵照上文所概述的全部程序,除了施加一級抗體溶液。使用數位彩色攝影機與ImagePro軟體(Media Cybernetics,Carlsbad,Calif)來擷取代表影像。針對三重染色的樣本,一次僅使用一個發射濾光片來拍攝各影像。接著使用Adobe Photoshop軟體(Adobe,San Jose,Calif)來製備分層合成影像(Layered montage)。 After immunostaining, the fluorescence is visualized using an appropriate fluorescent filter on an Olympus inverted epifluorescence microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented a fluorescent signal higher than that of the control stain, in which all procedures outlined above were followed, except for the application of primary antibody solution. Use a digital color camera and ImagePro software (Media Cybernetics, Carlsbad, Calif) to capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop software (Adobe, San Jose, Calif) to prepare layered composite images (Layered montage).
修改的伍德柏-布萊克規程(A):在於此神經誘導組成物中培養之後,所有細胞型即轉形成具有雙極形態及伸長突起之細胞。亦觀察到其他更大的非雙極形態。此外,所誘導之細胞群為巢蛋白染色陽性,巢蛋白為多潛能神經幹細胞與前驅細胞之標記。 Modified Woodbury-Black protocol (A) : After culturing in this nerve-inducing composition, all cell types are transformed into cells with bipolar morphology and elongated processes. Other larger non-bipolar patterns have also been observed. In addition, the induced cell population was positive for nestin staining, and nestin was a marker for pluripotent neural stem cells and precursor cells.
修改的伍德柏-布萊克規程(B):當在組織培養塑膠(TCP)盤上重複時,未觀察到巢蛋白表現,除非將層黏蛋白預吸附至 培養物表面。為進一步評估巢蛋白表現細胞是否可隨後繼續產生成熟神經元,將PPDC與纖維母細胞暴露於NPE+RA(1μM)中,RA為已知可誘導神經幹細胞與前驅細胞分化成此類細胞(2,3,4)之培養基組成物。將細胞針對TuJ1(不成熟與成熟神經元之標記)、GFAP(星狀細胞之標記)、及巢蛋白進行染色。在此類條件下未偵測出TuJ1,也未觀察到具有神經元形態之細胞。此外,PPDC不再表現巢蛋白及GF AP,如藉由免疫細胞化學所判定。 Modified Woodbury-Black protocol (B) : When repeated on a tissue culture plastic (TCP) plate, no nestin performance was observed, unless laminin was pre-adsorbed to the culture surface. To further assess whether nestin-expressing cells can subsequently continue to produce mature neurons, PPDC and fibroblasts were exposed to NPE + RA (1 μM), which is known to induce neural stem cells and precursor cells to differentiate into such cells (2 , 3, 4) medium composition. The cells were stained for TuJ1 (marker of immature and mature neurons), GFAP (marker of stellate cells), and nestin. TuJ1 was not detected under such conditions, and cells with neuronal morphology were not observed. In addition, PPDC no longer expressed nestin and GF AP, as determined by immunocytochemistry.
兩階段分化:將臍及胎盤PPDC分離株(以及分別作為陰性及陽性對照細胞型之人類纖維母細胞及嚙齒動物神經前驅)接種於經層黏蛋白(神經促進)塗布之盤上,並暴露於已知促進神經前驅分化成神經元及星狀細胞之13種不同生長條件(及兩種對照條件)。此外,添加兩種條件以檢測GDF5與BMP7對PPDC分化之影響。一般而言,採取兩步驟分化方式,其中首先將細胞放置於神經前驅擴增條件中歷時6天之時期,接著放置於全分化條件歷時7天。在形態學上,臍與胎盤衍生細胞兩者在此程序之整個時程皆展現細胞形態之基本改變。然而,未觀察到神經元或星狀細胞形狀之細胞,除了在對照組、神經前驅接種之條件中。免疫細胞化學(對人類巢蛋白、TuJ1、與GFAP為陰性)確認形態學之觀察。 Two-stage differentiation: inoculate the umbilical and placental PPDC isolates (and human fibroblasts and rodent neural precursors as negative and positive control cell types, respectively) on discs coated with laminin (neuropromotion) and exposed to Thirteen different growth conditions (and two control conditions) known to promote neural precursor differentiation into neurons and stellate cells are known. In addition, two conditions were added to examine the effect of GDF5 and BMP7 on PPDC differentiation. In general, a two-step differentiation method is adopted, in which cells are first placed in a neural precursor expansion condition for a period of 6 days, and then placed in a fully differentiated condition for 7 days. Morphologically, both umbilical and placental-derived cells exhibit basic changes in cell morphology throughout the course of this procedure. However, cells in the shape of neurons or stellate cells were not observed, except in the conditions of control group, nerve precursor inoculation. Immunocytochemistry (negative to human nestin, TuJ1, and GFAP) confirmed morphological observations.
多重生長因子:在暴露於各種神經分化劑一週之後,將細胞針對指示神經前驅(人類巢蛋白)、神經元(TuJ1)、及星狀細胞(GFAP)之標記進行染色。在第一階段中生長於不含血清之培養基的細胞具有與該些含血清(2%或10%)之培養基中的細胞不同之形 態,指出潛在的神經分化。特定言之,在將臍衍生細胞暴露於EGF與bFGF,接著暴露於SHH、FGF8、GDNF、BDNF、與視黃酸之兩步程序之後,細胞顯示類似於所培養的星狀細胞之形態的伸長突起。當2% FBS或10% FBS包括在分化之第一階段中時,細胞數目增加且細胞形態與高密度對照組培養物無異。人類巢蛋白、TuJ1、或GFAP的免疫細胞化學分析未證明潛在的神經分化。 Multiple growth factors: After one week of exposure to various neural differentiation agents, cells were stained for markers indicating neural precursors (human nestin), neurons (TuJ1), and stellate cells (GFAP). The cells grown in the serum-free medium in the first stage have a different form from those in the serum-containing medium (2% or 10%), indicating potential neural differentiation. Specifically, after exposing umbilical-derived cells to EGF and bFGF, followed by two-step procedures of SHH, FGF8, GDNF, BDNF, and retinoic acid, the cells showed elongation similar to the cultured stellate cells Protruding. When 2% FBS or 10% FBS was included in the first stage of differentiation, the cell number increased and the cell morphology was no different from the high-density control culture. Immunocytochemical analysis of human nestin, TuJ1, or GFAP did not demonstrate potential neural differentiation.
神經前驅與PPDC共培養:PPDC係經接種至兩天前接種於神經擴增條件(NPE+F+E)中的大鼠神經前驅之培養物上。儘管經接種之PPDC之視覺確認證明這些細胞係以單一細胞接種,但是接種後4天(總計6天)的人類特異性核染色(hNuc)顯示,其趨向於捲成球狀(ball up)且避免與神經前驅接觸。此外,當PPDC附著時,這些細胞展開且似乎受到大鼠來源的分化神經元之神經支配,表示PPDC可能已分化成肌細胞。此觀察係基於在相位差顯微鏡下之形態。另一觀察係一般大細胞體(大於神經前驅)擁有形似神經前驅之形態,具有細突起沿多個方向跨出。hNuc染色(見於一半的細胞核)顯示,在一些情況中,這些人類細胞可與大鼠前驅融合,且假定(assume)它們的表型。僅含有神經前驅之對照組孔相較於含有臍或胎盤PPDC之共培養孔,具有較少總前驅及明顯分化之細胞,此進一步表示,臍與胎盤衍生細胞兩者均影響神經前驅之分化及行為,其作用係藉由釋放趨化激素與細胞介素,或藉由接觸媒介作用。 Co-culture of neural precursors and PPDC : PPDC was inoculated to the culture of rat neural precursors in neural expansion conditions (NPE + F + E) two days before inoculation. Although visual confirmation of the inoculated PPDC proved that these cell lines were inoculated with a single cell, human specific nuclear staining (hNuc) 4 days after inoculation (total 6 days) showed that it tended to ball up and Avoid contact with neural precursors. In addition, when PPDC is attached, these cells expand and appear to be innervated by rat-derived differentiated neurons, indicating that PPDC may have differentiated into myocytes. This observation is based on the morphology under a phase contrast microscope. Another observation is that the general large cell body (larger than the neural precursor) has a shape resembling a neural precursor, with fine protrusions that span in multiple directions. hNuc staining (found in half of the nucleus) shows that in some cases, these human cells can be fused with rat precursors and assume their phenotype. Control wells containing only neural precursors have fewer total precursors and significantly differentiated cells than co-culture wells containing umbilical or placental PPDC, which further indicates that both umbilical and placental-derived cells affect the differentiation of neural precursors and Behavior, its role is through the release of chemokines and cytokines, or through contact with mediators.
總結:進行多個規程以判定PPDC分化成神經譜系細胞之短期潛能。這些包括形態學之相位差成像與針對巢蛋白、TuJ1、及GFAP(分別為與多潛能神經幹細胞及前驅細胞、不成熟及成熟神經元、及星狀細胞相關聯的蛋白質)之免疫細胞化學之組合。 Summary: Several procedures were performed to determine the short-term potential of PPDC to differentiate into neural lineage cells. These include morphological phase contrast imaging and immunocytochemistry for nestin, TuJ1, and GFAP (proteins associated with pluripotent neural stem cells and precursor cells, immature and mature neurons, and stellate cells, respectively) combination.
評估臍與胎盤衍生細胞(統稱為產後衍生細胞或PPDC)進行長期分化成神經譜系細胞之能力。 To evaluate the ability of umbilical and placental-derived cells (collectively called postpartum-derived cells or PPDC) to differentiate into neural lineage cells over a long period of time.
PPDC之分離及擴增:如先前實例所述將PPDC分離並擴增。 Isolation and amplification of PPDC : Isolate and amplify PPDC as described in the previous example.
PPDC細胞解凍及接種:將先前生長於生長培養基中之PPDC冷凍等分(臍(022803)P11;(042203)P11;(071003)P12;胎盤(101503)P7)解凍且以5,000個細胞/cm2接種於經層黏蛋白(BD,Franklin Lakes,N.J.)塗布之T-75培養瓶中之含有B27(B27補充物,Invitrogen)、L-麩醯胺酸(4mM)、及青黴素/鏈黴素(10毫升)之Neurobasal-A培養基(Invitrogen,Carlsbad,Calif.),其組合在本文中稱為神經前驅擴增(NPE)培養基。將NPE培養基用bFGF(20奈克/毫升,Peprotech,Rocky Hill,N.J.)及EGF(20奈克/毫升, Peprotech,Rocky Hill,N.J.)進一步補充,本文稱為NPE+bFGF+EGF。 Thawing and inoculation of PPDC cells: Frozen aliquots of PPDC previously grown in growth medium (umbilical (022803) P11; (042203) P11; (071003) P12; placenta (101503) P7) were thawed and inoculated at 5,000 cells / cm2 The T-75 culture flask coated with laminin (BD, Franklin Lakes, NJ) contains B27 (B27 supplement, Invitrogen), L-glutamic acid (4 mM), and penicillin / streptomycin (10 Ml) of Neurobasal-A medium (Invitrogen, Carlsbad, Calif.), The combination of which is referred to herein as a neural precursor expansion (NPE) medium. The NPE medium is further supplemented with bFGF (20 ng / ml, Peprotech, Rocky Hill, NJ) and EGF (20 ng / ml, Peprotech, Rocky Hill, NJ), which is referred to herein as NPE + bFGF + EGF.
對照組細胞接種:此外,將成體人類皮膚纖維母細胞(P11,Cambrex,Walkersville,Md.)及間葉幹細胞(P5,Cambrex)解凍並以相同細胞接種密度接種於經層黏蛋白塗布之T-75培養瓶上之NPE+bFGF+EGF中。作為進一步對照組,使纖維母細胞、臍與胎盤PPDC生長於生長培養基中歷時所有培養物所指定的時期。 Control cell inoculation: In addition, adult human skin fibroblasts (P11, Cambrex, Walkersville, Md.) And mesenchymal stem cells (P5, Cambrex) were thawed and inoculated with laminin-coated T at the same cell seeding density -75 NPE + bFGF + EGF on culture flask. As a further control group, fibroblasts, navel, and placental PPDC were grown in growth medium for the period specified by all cultures.
細胞擴增:每週將所有培養物之培養基用新鮮培養基替換一次,且觀察細胞之擴增。大致上,各培養物由於在NPE+bFGF+EGF中生長受限,在一個月期間內僅繼代一次。 Cell expansion: Replace the culture medium of all cultures with fresh medium once a week, and observe the expansion of the cells. In general, each culture was only subcultured once in a month due to restricted growth in NPE + bFGF + EGF.
免疫細胞化學:在一個月期間之後,將所有培養瓶在室溫下用冷4%(w/v)多聚甲醛(Sigma)固定10分鐘。免疫細胞化學係使用針對下列者的抗體來執行:TuJ1(BIII Tubulin;1:500;Sigma,St.Louis,Mo.)及GFAP(膠細胞纖維酸性蛋白質;1:2000;DakoCytomation,Carpinteria,Calif.)。簡言之,用磷酸鹽緩衝液(PBS)清洗培養物然後將其暴露於含PBS、4%(v/v)山羊血清(Chemic on,Temecula,Calif.)與0.3%(v/v)Triton(Triton X-100;Sigma)的蛋白質阻斷液中30分鐘以獲取細胞內抗原。接著在室溫下將一級抗體(稀釋於阻斷液中)施用於培養物歷時1小時。接著,將一級抗體溶液移除,用PBS清洗培養物,然後施用二級抗體溶液(在室溫下1小時),該二級抗體溶液含有阻斷劑以及山羊抗小鼠IgG--Texas Red(1:250;Molecular Probes,Eugene,OR)及山羊抗兔IgG-Alexa 488 (1:250;Molecular Probes)。隨後清洗培養物,然後施用10微莫耳DAPI(Molecular Probes)10分鐘以可視化細胞核。 Immunocytochemistry: After a one-month period, all culture flasks were fixed with cold 4% (w / v) paraformaldehyde (Sigma) at room temperature for 10 minutes. The Department of Immunocytochemistry was performed using antibodies against: TuJ1 (BIII Tubulin; 1: 500; Sigma, St. Louis, Mo.) and GFAP (Glial Fibrillary Acidic Protein; 1: 2000; DakoCytomation, Carpinteria, Calif. ). Briefly, the culture was washed with phosphate buffered saline (PBS) and then exposed to PBS, 4% (v / v) goat serum (Chemic on, Temecula, Calif.) And 0.3% (v / v) Triton (Triton X-100; Sigma) protein blocking solution for 30 minutes to obtain intracellular antigens. The primary antibody (diluted in blocking solution) was then applied to the culture at room temperature for 1 hour. Next, the primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking agent and goat anti-mouse IgG--Texas Red ( 1: 250; Molecular Probes, Eugene, OR) and goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes). The culture was subsequently washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.
在免疫染色後,在Olympus倒立落射螢光顯微鏡(Olympus,Melville,N.Y.)上使用適當螢光濾光片來可視化螢光。在所有情況下,陽性染色代表高於對照組染色的螢光訊號,其中遵照上文所概述的全部程序,除了施加一級抗體溶液。使用數位彩色攝影機與ImagePro軟體(Media Cybernetics,Carlsbad,Calif.)來擷取代表影像。針對三重染色的樣本,一次僅使用一個發射濾光片來拍攝各影像。接著使用Adobe Photoshop軟體(Adobe,San Jose,Calif.)來製備分層合成影像(Layered montage)。 After immunostaining, the fluorescence is visualized using an appropriate fluorescent filter on an Olympus inverted epifluorescence microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented a fluorescent signal higher than that of the control stain, in which all procedures outlined above were followed, except for the application of primary antibody solution. Use a digital color camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.) To capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop software (Adobe, San Jose, Calif.) To prepare layered composite images (Layered montage).
NPE+bFGF+EGF培養基延緩PPDC之增生且更改其形態。在接種之後,一亞群之PPDC立即附著至經層黏蛋白塗布之培養瓶。此可能因為細胞隨冷凍/解凍過程變化而死亡或由於新的生長條件。附著的細胞採用不同於在生長培養基中所觀察到的細胞之形態。 NPE + bFGF + EGF medium delays the proliferation of PPDC and changes its morphology. Immediately after inoculation, a subset of PPDC was attached to the culture flask coated with laminin. This may be due to cell death due to changes in the freezing / thawing process or due to new growth conditions. The attached cells adopt a morphology different from that observed in the growth medium.
臍衍生細胞之殖株表現神經元蛋白質:培養物係於解凍/接種後一個月固定且針對神經元蛋白質TuJ1及GFAP(在星狀細 胞中發現之中間絲)進行染色。儘管發現生長於生長培養基中之所有對照組培養物及生長於NPE+bFGF+EGF培養基中之人類纖維母細胞及MSC為TuJ1-/GFAP-,但是在臍與胎盤PPDC中偵測出TuJ1。在具有及不具有類神經元形態之細胞中觀察到表現。所有培養物中皆未觀察到GFAP之表現。表現TuJ1且具有類神經元形態之細胞的百分比小於或等於總族群之1%(n=3個經測試的臍衍生細胞分離株)。儘管未定量,但是TuJ1+且不具有神經元形態之細胞在臍衍生細胞培養物中之百分比高於在胎盤衍生細胞培養物中之百分比。這些結果似乎具有特異性,因為生長培養基中之年齡匹配的對照組不表現TuJ1。 Umbilical-derived cell colonies express neuronal proteins: cultures were fixed one month after thawing / inoculation and stained for neuronal proteins TuJ1 and GFAP (intermediate filaments found in stellate cells). Although all control cultures grown in growth medium and human fibroblasts and MSCs grown in NPE + bFGF + EGF medium were found to be TuJ1- / GFAP-, TuJ1 was detected in umbilical and placental PPDC. Performance is observed in cells with and without neuron-like morphology. No performance of GFAP was observed in all cultures. The percentage of cells exhibiting TuJ1 and having neuron-like morphology is less than or equal to 1% of the total population (n = 3 tested umbilical-derived cell isolates). Although not quantified, the percentage of TuJ1 + cells without neuronal morphology in umbilical derived cell culture is higher than in placental derived cell culture. These results seem to be specific because the age-matched control group in growth medium does not exhibit TuJ1.
總結:用於自臍衍生細胞產生經分化之神經元(基於TuJ1表現及神經元形態)之方法係經發展。儘管早於一個月的體外培養未檢測出TuJ1之表現,但是很明顯,至少一小族群的臍衍生細胞可經由預設分化(default differentiation)或經由在暴露於補充有L-麩醯胺酸、鹼性FGF、與EGF之最低培養基一個月的長期誘導而形成神經元。 Summary: Methods for generating differentiated neurons (based on TuJ1 performance and neuronal morphology) from umbilical derived cells have been developed. Although TuJ1 performance was not detected in vitro cultures older than one month, it is clear that at least a small group of umbilical-derived cells can undergo default differentiation or through exposure to supplemented with L-glutamine, The minimal medium of basic FGF and EGF is induced for a month to form neurons.
臍與胎盤衍生細胞(統稱為產後衍生細胞或PPDC)經由非接觸依賴性(營養)機制對成體神經幹細胞與前驅細胞存活及分化之影響係經檢測。 The effects of umbilical and placental-derived cells (collectively called postpartum-derived cells or PPDCs) on the survival and differentiation of adult neural stem cells and precursor cells through non-contact-dependent (nutrition) mechanisms have been tested.
成體神經幹細胞及前驅細胞分離:Fisher 344成鼠係藉由CO2窒息接著頸椎脫位術犧牲。使用骨鉗將全部大腦完整移除,且基於在大腦之運動及體感覺區域之後的冠狀切口將海馬迴組織解剖(Paxinos,G.& Watson,C.1997.The Rat Brain in Stereotaxic Coordinates)。將組織於Neurobasal-A培養基(Invitrogen,Carlsbad,Calif.)中清洗,該Neurobasal-A培養基含有B27(B27補充物;Invitrogen)、L-麩醯胺酸(4mM;Invitrogen)、及青黴素/鏈黴素(Invitrogen),其等之組合在本文中稱為神經前驅擴增(NPE)培養基。將NPE培養基用bFGF(20奈克/毫升,Peprotech,Rocky Hill,N.J.)及EGF(20奈克/毫升,Peprotech,Rocky Hill,N.J.)進一步補充,本文稱為NPE+bFGF+EGF。 Isolation of adult neural stem cells and precursor cells: Fisher 344 adult rats were sacrificed by CO 2 asphyxia followed by cervical dislocation. All brains were completely removed using bone forceps, and hippocampal anatomy was dissected based on a coronal incision after the movement and somatosensory areas of the brain (Paxinos, G. & Watson, C. 1997. The Rat Brain in Stereotaxic Coordinates). Tissues were washed in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.) Containing B27 (B27 supplement; Invitrogen), L-glutamic acid (4 mM; Invitrogen), and penicillin / streptomyces Invitrogen, and the like, are referred to herein as neural precursor expansion (NPE) medium. The NPE medium is further supplemented with bFGF (20 ng / ml, Peprotech, Rocky Hill, NJ) and EGF (20 ng / ml, Peprotech, Rocky Hill, NJ), which is referred to herein as NPE + bFGF + EGF.
在清洗之後,移除上覆腦膜,且將組織用解剖刀切碎。收集切碎之組織且添加75%總體積之胰蛋白酶/EDTA(Invitrogen)。亦添加DNase(100微升/8毫升總體積,Sigma,St.Louis,Mo.)。接著,使組織/培養基依序通過18號針頭、20號針頭、與最終25號針頭各一次(所有針頭皆來自Becton Dickinson,Franklin Lakes,N.J.)。將混合物在250g下離心3分鐘。移除上清液,添加新鮮NPE+bFGF+EGF且將團塊再懸浮。使所得細胞懸浮液通過40微米細胞濾器(Becton Dickinson),接種於經層黏蛋白塗布之T-75培養瓶(Becton Dickinson)或低團簇24孔盤(Becton Dickinson)上,且生 長於NPE+bFGF+EGF培養基中直到獲得概述研究所需之足夠細胞數目。 After washing, the overlying meninges are removed, and the tissue is minced with a scalpel. The minced tissue was collected and 75% of the total volume of trypsin / EDTA (Invitrogen) was added. DNase (100 μl / 8 ml total volume, Sigma, St. Louis, Mo.) was also added. Next, the tissue / medium was sequentially passed through the 18-gauge needle, the 20-gauge needle, and finally the 25-gauge needle (all needles were from Becton Dickinson, Franklin Lakes, N.J.). The mixture was centrifuged at 250g for 3 minutes. The supernatant was removed, fresh NPE + bFGF + EGF was added and the pellet was resuspended. The resulting cell suspension was passed through a 40 micron cell strainer (Becton Dickinson), seeded on a T-75 culture flask (Becton Dickinson) coated with laminin or a low-clustered 24-well dish (Becton Dickinson), and grown on NPE bFGF + EGF medium until the sufficient number of cells required for the summary study is obtained.
PPDC接種:先前生長於生長培養基中之產後衍生細胞(臍(022803)P12、(042103)P12、(071003)P12;胎盤(042203)P12)係以5,000個細胞/跨孔插入物(transwell insert)(適用24孔盤之大小)接種且於生長培養基中於插入物中生長一週之時期以達成長滿。 PPDC inoculation: postnatally derived cells (umbilical (022803) P12, (042103) P12, (071003) P12; placenta (042203) P12) previously grown in growth medium with 5,000 cells / transwell insert (Applies to the size of a 24-well plate) Inoculate and grow in the insert in the growth medium for a period of one week to achieve fullness.
成體神經前驅接種:生長為神經球或單一細胞之神經前驅係以大致2,000個細胞/孔之密度接種至經層黏蛋白塗布之24孔盤上之NPE+bFGF+EGF中歷時一天之時期以促進細胞附著。一天後,根據以下方案添加含有產後細胞之跨孔插入物: Adult nerve precursor inoculation: a nerve precursor that grows as a neurosphere or single cell is inoculated at a density of approximately 2,000 cells / well into NPE + bFGF + EGF on a 24-well plate coated with laminin for a period of one day to Promote cell attachment. One day later, add a transwell insert containing postpartum cells according to the following protocol:
a. 跨孔(臍衍生細胞於生長培養基中,200微升)+神經前驅(NPE+bFGF+EGF,1毫升) a. Cross-well (umbilical-derived cells in growth medium, 200 μl) + neural precursor (NPE + bFGF + EGF, 1 ml)
b. 跨孔(胎盤衍生細胞於生長培養基中,200微升)+神經前驅(NPE+bFGF+EGF,1毫升) b. Trans-well (placenta-derived cells in growth medium, 200 μl) + neural precursor (NPE + bFGF + EGF, 1 ml)
c. 跨孔(成體人類皮膚纖維母細胞[1 F 1853;Cambrex,Walkersville,Md.]P12於生長培養基中,200微升)+神經前驅(NPE+bFGF+EGF,1毫升) c. Transwell (adult human skin fibroblasts [1 F 1853; Cambrex, Walkersville, Md.] P12 in growth medium, 200 μl) + neural precursors (NPE + bFGF + EGF, 1 ml)
d. 對照組:單獨神經前驅(NPE+bFGF+EGF,1毫升) d. Control group: neural precursor alone (NPE + bFGF + EGF, 1 ml)
e. 對照組:單獨神經前驅(僅NPE,1毫升) e. Control group: nerve precursor alone (NPE only, 1 ml)
免疫細胞化學:在共培養物中7天之後,將所有條件在室溫下用冷4%(w/v)多聚甲醛(Sigma)固定10分鐘之時期。使用針對表14-1中所列舉之表位的抗體來執行免疫細胞化學。簡言之, 用磷酸鹽緩衝液(PBS)清洗培養物然後將其暴露於含PBS、4%(v/v)山羊血清(Chemic on,Temecula,Calif.)與0.3%(v/v)Triton(Triton X-100;Sigma)的蛋白質阻斷液中30分鐘以獲取細胞內抗原。接著在室溫下將一級抗體(稀釋於阻斷液中)施用於培養物歷時1小時。接著,將一級抗體溶液移除,用PBS清洗培養物,然後施用二級抗體溶液(在室溫下1小時),該二級抗體溶液含有阻斷溶液以及山羊抗小鼠IgG--Texas Red(1:250;Molecular Probes,Eugene,OR)及山羊抗兔IgG-Alexa 488(1:250;Molecular Probes)。隨後清洗培養物,然後施用10微莫耳DAPI(Molecular Probes)10分鐘以可視化細胞核。 Immunocytochemistry: After 7 days in the co-culture, all conditions were fixed with cold 4% (w / v) paraformaldehyde (Sigma) for 10 minutes at room temperature. Immunocytochemistry was performed using antibodies against the epitopes listed in Table 14-1. Briefly, the culture was washed with phosphate buffered saline (PBS) and then exposed to PBS, 4% (v / v) goat serum (Chemic on, Temecula, Calif.) And 0.3% (v / v) Triton (Triton X-100; Sigma) protein blocking solution for 30 minutes to obtain intracellular antigens. The primary antibody (diluted in blocking solution) was then applied to the culture at room temperature for 1 hour. Next, the primary antibody solution was removed, the culture was washed with PBS, and then a secondary antibody solution (1 hour at room temperature) was applied. The secondary antibody solution contained a blocking solution and goat anti-mouse IgG--Texas Red ( 1: 250; Molecular Probes, Eugene, OR) and goat anti-rabbit IgG-Alexa 488 (1: 250; Molecular Probes). The culture was subsequently washed, and then 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize the nucleus.
在免疫染色後,在Olympus倒立落射螢光顯微鏡(Olympus,Melville,N.Y.)上使用適當螢光濾光片來可視化螢光。在所有情況下,陽性染色代表高於對照組染色的螢光訊號,其中遵照上文所概述的全部程序,除了施加一級抗體溶液。使用數位彩色攝影機與ImagePro軟體(Media Cybernetics,Carlsbad,Calif.)來擷取代表影像。針對三重染色的樣本,一次僅使用一個發射濾光片來拍攝各影像。接著使用Adobe Photoshop軟體(Adobe,San Jose,Calif.)來製備分層合成影像(Layered montage)。 After immunostaining, the fluorescence is visualized using an appropriate fluorescent filter on an Olympus inverted epifluorescence microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented a fluorescent signal higher than that of the control stain, in which all procedures outlined above were followed, except for the application of primary antibody solution. Use a digital color camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.) To capture the table image. For triple-stained samples, only one emission filter was used to shoot each image at a time. Then use Adobe Photoshop software (Adobe, San Jose, Calif.) To prepare layered composite images (Layered montage).
神經前驅分化之定量分析:海馬迴神經前驅分化之定量係經檢測。每個條件計數最少1000個細胞,或若更少,則計數該條件中所觀察到的細胞總數。對於給定染色劑為陽性的細胞百分比係藉由將陽性細胞數目除以藉由DAPI(核)染色所判定之細胞總數來評估。 Quantitative analysis of neural precursor differentiation: Quantitative analysis of hippocampal gyrus neural precursor differentiation has been tested. Each condition counts a minimum of 1000 cells, or if fewer, counts the total number of cells observed in that condition. The percentage of cells that are positive for a given stain is evaluated by dividing the number of positive cells by the total number of cells determined by DAPI (nuclear) staining.
質譜分析及2D凝膠電泳:為了識別獨特的、因共培養而分泌之因子,將在培養物固定之前所取得的條件培養基樣本在-80℃下冷凍過夜。隨後將樣本施加至超濾自旋裝置(截留MW為30kD)。將阻留物(retentate)施加至免疫親和層析法(抗Hu白蛋白;IgY)(免疫親和性不會將白蛋白自樣本移除)。藉由MALDI分析濾液。將通過液(pass through)施加至Cibachron Blue親和層析法。藉由SDS-PAGE及2D凝膠電泳分析樣本。 Mass spectrometry and 2D gel electrophoresis: In order to identify unique factors secreted by co-cultivation, samples of the conditioned medium obtained before the culture was fixed were frozen at -80 ° C overnight. The sample was then applied to an ultrafiltration spin device (with a cut-off MW of 30 kD). Retentate is applied to immunoaffinity chromatography (anti-Hu albumin; IgY) (immunoaffinity does not remove albumin from the sample). The filtrate was analyzed by MALDI. Pass through was applied to Cibachron Blue affinity chromatography. The samples were analyzed by SDS-PAGE and 2D gel electrophoresis.
PPDC共培養刺激成體神經前驅分化:在與臍或胎盤衍生細胞培養之後,經共培養之衍生自成鼠海馬迴之神經前驅細胞展現沿著中樞神經系統中所有三大譜系的顯著分化。在共培養五天之後清楚觀察到此作用,其中許多細胞發展出複雜突起且喪失屬於前驅細胞分裂表徵之相明亮特徵。相反地,在bFGF及EGF不存在下單獨生長之神經前驅外觀不健康且存活受限。 PPDC co-culture stimulates adult neural precursor differentiation: after culturing with umbilical or placental-derived cells, co-cultured neural precursor cells derived from adult rat hippocampus exhibit significant differentiation along all three major lineages in the central nervous system. This effect was clearly observed after five days of co-cultivation, in which many cells developed complex processes and lost the bright features that are characteristic of precursor cell division. Conversely, neural precursors that grow alone in the absence of bFGF and EGF appear unhealthy and have limited survival.
在程序完成之後,將培養物針對指示未分化幹細胞及前驅細胞(巢蛋白)、不成熟及成熟神經元(TuJ1)、星狀細胞(GFAP)、及成熟寡樹突細胞(MBP)之標記進行染色。沿著所有三種譜系之分化係經確認,儘管對照組條件並未展現顯著分化,如大部分細胞保留巢蛋白陽性染色所證明。儘管臍及胎盤衍生細胞皆誘導細胞分化,但是與胎盤衍生細胞之共培養物中的所有三種譜系之分化程度小於與臍衍生細胞之共培養物中。 After the procedure is completed, the culture is targeted for markers indicating undifferentiated stem cells and precursor cells (nestin), immature and mature neurons (TuJ1), stellate cells (GFAP), and mature oligodendritic cells (MBP) dyeing. The differentiation lines along all three lineages were confirmed, although the control conditions did not show significant differentiation, as evidenced by the majority of cells retaining nestin-positive staining. Although both umbilical and placenta-derived cells induce cell differentiation, all three lineages in co-cultures with placenta-derived cells are less differentiated than in co-cultures with umbilical-derived cells.
與臍衍生細胞共培養之後的分化神經前驅之百分比係經定量(表24-2)。臍衍生細胞顯著提高成熟寡樹突細胞(MBP)之數目(24.0%相較於兩種對照組條件之0%)。此外,共培養提高培養物中GFAP+星狀細胞與TuJ1+神經元的數目(分別為47.2%與8.7%)。這些結果係經巢蛋白染色之確認,指示在共培養之後喪失前驅狀態(13.4%相較於對照組條件4之71.4%)。 The percentage of differentiated neural precursors after co-culture with umbilical derived cells was quantified (Table 24-2). Umbilical-derived cells significantly increased the number of mature oligodendrocytes (MBP) (24.0% compared to 0% for both control conditions). In addition, co-cultivation increased the number of GFAP + stellate cells and TuJ1 + neurons in the culture (47.2% and 8.7%, respectively). These results were confirmed by nestin staining, indicating the loss of the precursor state after co-cultivation (13.4% compared to 71.4% of control group condition 4).
雖然分化似乎亦受成體人類纖維母細胞影響,但是此類細胞無法促進成熟寡樹突細胞之分化,也不能產生可察覺量的神經元。儘管未定量,但是纖維母細胞似乎確實增強神經前驅之生存。 Although differentiation also appears to be affected by adult human fibroblasts, such cells cannot promote the differentiation of mature oligodendrocytes or produce appreciable amounts of neurons. Although not quantified, fibroblasts do seem to enhance the survival of neural precursors.
獨特化合物之識別:檢測來自臍及胎盤衍生共培養物之條件培養基,與適當對照組(NPE培養基±I.7%血清,來自與纖維母細胞之共培養的培養基)之差異。潛在之獨特化合物係經識別且自其各自2D凝膠切除。 Identification of unique compounds: The difference between the conditioned medium derived from the umbilical and placenta-derived co-cultures and the appropriate control group (NPE medium ± 1.7% serum, co-cultured with fibroblasts). Potentially unique compounds are identified and excised from their respective 2D gels.
總結:成體神經前驅細胞與臍或胎盤PPDC之共培養導致該些細胞之分化。此實例中所表現的結果指示,在與臍衍生細胞共培養之後,成體神經前驅細胞之分化尤其突出。特定言之,在臍衍生細胞之共培養物中產生顯著百分比的成熟寡樹突細胞。 Summary: Co-culture of adult neural precursor cells with umbilical or placental PPDC leads to the differentiation of these cells. The results presented in this example indicate that after co-cultivation with umbilical derived cells, the differentiation of adult neural precursor cells is particularly prominent. In particular, a significant percentage of mature oligodendritic cells are produced in co-cultures of umbilical derived cells.
衍生自產後臍及胎盤之細胞可用於再生性治療。對藉由用可生物降解材料將產後衍生細胞(PPDC)移植至SCID小鼠中所生產之組織進行評估。所評估之材料為Vicryl非織物、35/65 PCL/PGA發泡體、及RAD 16自組裝肽水凝膠。 Cells derived from the postpartum umbilical and placental can be used for regenerative treatment. The tissues produced by transplanting postpartum-derived cells (PPDC) into SCID mice with biodegradable materials were evaluated. The materials evaluated were Vicryl non-woven fabric, 35/65 PCL / PGA foam, and RAD 16 self-assembled peptide hydrogel.
細胞培養:將胎盤衍生細胞及臍衍生細胞生長於生長培養基(DMEM-低葡萄糖(Gibco,Carlsbad Calif.)、15%(v/v)胎牛血清(Cat.#SH30070.03;Hyclone,Logan,Utah)、0.001%(v/v)β-巰基乙醇(Sigma,St.Louis,Mo.)、青黴素/鏈黴素(Gibco)),該生長培養基係在經明膠塗布之培養瓶中。 Cell culture: grow placenta-derived cells and umbilical-derived cells in growth medium (DMEM-low glucose (Gibco, Carlsbad Calif.), 15% (v / v) fetal bovine serum (Cat. # SH30070.03; Hyclone, Logan, Utah), 0.001% (v / v) β-mercaptoethanol (Sigma, St. Louis, Mo.), penicillin / streptomycin (Gibco)), the growth medium is in a gelatin-coated culture flask.
樣本製備:將一百萬個存活細胞接種於15微升生長培養基中至5mm直徑、2.25mm厚Vicryl非織物支架(64.33毫克/cc;批號3547-47-1)或5mm直徑35/65 PCL/PGA發泡體(批號3415-53)上。使細胞附著兩小時,之後添加更多生長培養基以覆蓋支架。使細胞於支架上生長隔夜。亦將不具有細胞的支架培養於培養基中。 Sample preparation: Inoculate one million viable cells in 15 microliters of growth medium to 5 mm diameter, 2.25 mm thick Vicryl non-woven scaffold (64.33 mg / cc; batch number 3547-47-1) or 5 mm diameter 35/65 PCL / PGA foam (Lot No. 3415-53). The cells were allowed to attach for two hours, after which more growth medium was added to cover the scaffold. The cells were grown on the scaffold overnight. The scaffold without cells was also cultured in the medium.
RAD16自組裝肽(3D Matrix,Cambridge,MA)係以無菌1%(w/v)於水中之溶液的形式獲得,以1:1混合其與1 x 106個細胞於10%(w/v)蔗糖(Sigma,St Louis,Mo.)、10mM HEPES於達爾伯克改良培養基(DMEM;Gibco),之後立即使用。細胞於RAD 16水凝膠中之最終濃度為1×106個細胞/100微升。 RAD16 self-assembling peptides (3D Matrix, Cambridge, MA) based on the form of a solution in water of the obtained in sterile 1% (w / v), to 1: 1 mixed with 1 x 10 6 cells in 10% (w / v ) Sucrose (Sigma, St Louis, Mo.), 10 mM HEPES in Dulbecco's modified medium (DMEM; Gibco), and used immediately thereafter. The final concentration of cells in the RAD 16 hydrogel was 1 × 10 6 cells / 100 μl.
測試材料(N=4/Rx) Test material (N = 4 / Rx)
a. Vicryl非織物+1×106個臍衍生細胞 a. Vicryl non-woven fabric + 1 × 10 6 umbilical derived cells
b. 35/65 PCL/PGA發泡體+1×106個臍衍生細胞 b. 35/65 PCL / PGA foam + 1 × 10 6 umbilical derived cells
c. RAD 16自組裝肽+1×106個臍衍生細胞 c. RAD 16 self-assembling peptide + 1 × 10 6 umbilical derived cells
d. Vicryl非織物+1×106個胎盤衍生細胞 d. Vicryl non-woven fabric + 1 × 10 6 placenta-derived cells
e. 35/65 PCL/PGA發泡體+1×106個胎盤衍生細胞 e. 35/65 PCL / PGA foam + 1 × 10 6 placenta-derived cells
f. RAD 16自組裝肽+1×106個胎盤衍生細胞 f. RAD 16 self-assembling peptide + 1 × 10 6 placental-derived cells
g. 35/65 PCL/PGA發泡體 g. 35/65 PCL / PGA foam
h. Vicryl非織物 h. Vicryl non-woven
動物準備:根據動物福利法案(Animal Welfare Act)之現行規定操作及供養動物。藉由遵守動物福利法規(9 CFR)且符合 Guide for the Care and Use of Laboratory Animals,7th edition中所公布的現行標準來實現對上文公共法案之遵守。 Animal preparation: Operate and feed animals in accordance with the current regulations of the Animal Welfare Act. By complying with the Animal Welfare Regulations (9 CFR) and complying with the current standards published in the Guide for the Care and Use of Laboratory Animals, 7th edition, compliance with the above public laws is achieved.
小鼠(小家鼠(Mus Musculus))/Fox Chase SCID/雄性(Harlan Sprague Dawley,Inc.,Indianapolis,Ind.),5週齡:SCID小鼠之所有操作皆在通風櫥中進行。將小鼠個別稱重且用60毫克/kg KETASET(鹽酸氯胺酮,Aveco Co.,Inc.,Fort Dodge,Iowa)與10毫克/kg ROMPUN(xylazine,Mobay Corp.,Shawnee,Kans.)及鹽水之混合物進行腹膜內注射來麻醉。在導入麻醉之後,使用動物電剪將動物自背頸區域至背腰區域之整個背部的毛髮剃光。隨後將該區域用氯己定二乙酸鹽(chlorhexidine diacetate)刷洗,用醇潤洗,乾燥,然後用1%有效碘(available iodine)之帶碘化合物(iodophor)水溶液塗抹。將眼科軟膏施加至眼睛以預防在麻醉期間組織乾燥。 Mice ( Mus Musculus) / Fox Chase SCID / Male ( Harlan Sprague Dawley, Inc., Indianapolis, Ind. ), 5 weeks old: All operations of SCID mice are performed in a fume hood. The mice were individually weighed and used 60 mg / kg KETASET (ketamine hydrochloride, Aveco Co., Inc., Fort Dodge, Iowa) and 10 mg / kg ROMPUN (xylazine, Mobay Corp., Shawnee, Kans.) And saline. The mixture is anesthetized by intraperitoneal injection. After the introduction of anesthesia, the animal's entire back hair is shaved from the back neck area to the back waist area using animal clippers. The area was then scrubbed with chlorhexidine diacetate, rinsed with alcohol, dried, and then smeared with a 1% aqueous solution of iodophor with available iodine. Ophthalmic ointment is applied to the eyes to prevent tissue drying during anesthesia.
皮下移植技術:在小鼠背部做出四個皮膚切口,各大致1.0cm長。兩個頭側位點(cranial site)係橫向地定位於背外側胸腔、手觸摸肩胛骨下邊緣後方約5-mm之區域,其中一個在脊柱左側,另一個在脊柱右側。另外兩個位點係橫向地定位於後薦腰(caudal sacro-Iumbar)處、手觸摸髂骨崤後方約5-mm之臀肌區域,中線兩側各一。根據試驗計劃,將植入物隨機放置於這些位點中。使皮膚與下伏結締組織分離以製成小口袋,將植入物放入(或將RAD16注射入)切口後方約1-cm處。將適當測試材料植入至皮下空間。將皮膚切口用金屬夾封閉。 Subcutaneous transplantation technique: Make four skin incisions on the back of the mouse, each approximately 1.0 cm long. The two cranial sites are located laterally in the dorsal lateral thoracic cavity, with the hand touching the region about 5-mm behind the lower edge of the scapula, one on the left side of the spine and the other on the right side of the spine. The other two sites are positioned laterally at the caudal sacro-Iumbar, with the hand touching the gluteal muscle area about 5-mm behind the iliac bone, one on each side of the midline. According to the test plan, the implants were randomly placed in these sites. Separate the skin from the underlying connective tissue to make a small pocket, and place (or inject RAD16 into) the implant about 1-cm behind the incision. Implant the appropriate test material into the subcutaneous space. The skin incision was closed with a metal clip.
動物安置:在整個研究時程中,將小鼠個別安置於微型隔離籠中,溫度範圍為64℉至79℉,且相對濕度為30%至70%,並維持大致12小時光/12小時暗循環。將溫度及相對濕度盡可能地維持在所述範圍內。食物由經照射的Pico Mouse Chow 5058(Purina Co.)組成且無限制飲水。 Animal placement: During the entire study period, mice were individually housed in micro-isolated cages with a temperature range of 64 ° F to 79 ° F and a relative humidity of 30% to 70%, and maintained for approximately 12 hours of light / 12 hours of darkness cycle. Keep the temperature and relative humidity within the range as possible. Food consists of irradiated Pico Mouse Chow 5058 (Purina Co.) and unlimited drinking water.
小鼠在其指定期間藉由二氧化碳來進行安樂死。將皮下植入位點連同其上覆皮膚切除且冷凍以用於組織學研究。 The mice were euthanized with carbon dioxide during their designated period. The subcutaneous implantation site with its overlying skin was excised and frozen for histological study.
組織學:將切除皮膚連同植入物用10%中性緩衝福馬林(Richard-Allan Kalamazoo,Mich.)固定。將樣本連同上覆及相鄰組織由中間對切,使用常規方法用石臘處理及包埋切割表面。使用常規方法,藉由薄片切片機獲得五微米組織切片且用蘇木精與曙紅(Poly Scientific Bay Shore,N.Y.)染色。 Histology: The excised skin together with the implant was fixed with 10% neutral buffered formalin (Richard-Allan Kalamazoo, Mich.). Cut the sample together with the overlying and adjacent tissues from the middle, using conventional methods to treat and embed the cut surface with paraffin. Using conventional methods, five micron tissue sections were obtained by a thin slicer and stained with hematoxylin and eosin (Poly Scientific Bay Shore, N.Y.).
30天後,經植入SCID小鼠皮下之發泡體(無細胞)中有極小的內生長。相反地,含有臍衍生細胞或胎盤衍生細胞之植入發泡體中充滿廣泛的組織。於Vicryl非織物支架中觀察到一些組織內生長。接種臍或胎盤衍生細胞之非織物支架顯示基質沉積及成熟血管增加。 After 30 days, there was minimal ingrowth in the foam (cell-free) implanted under the skin of SCID mice. Conversely, implanted foam containing umbilical-derived cells or placental-derived cells is filled with extensive tissue. Some tissue growth was observed in Vicryl non-woven scaffold. Non-woven scaffolds seeded with umbilical or placental-derived cells show increased matrix deposition and mature blood vessels.
總結:合成性可吸收非織物/發泡體盤(5.0mm直徑×1.0mm厚)或自組裝肽水凝膠係與經衍生自人類臍或胎盤的細胞 接種,且雙側皮下植入於SCID小鼠之背脊部。結果證明,產後衍生細胞可大幅增加可生物降解支架中高品質的組織形成。 Summary: Synthetic absorbable non-woven / foam discs (5.0mm diameter x 1.0mm thick) or self-assembling peptide hydrogels are inoculated with cells derived from human umbilicus or placenta and implanted bilaterally subcutaneously in SCID The back of the mouse. The results prove that postpartum-derived cells can significantly increase the formation of high-quality tissue in biodegradable scaffolds.
端粒酶的功能是合成端粒重複序列(telomere repeat),端粒重複序列用來保護染色體完整性並延長細胞的複製壽命(Liu,K,等人,PNAS,1999;96:5147-5152)。端粒酶由兩個組分所組成,即端粒酶RNA模板(hTER)與端粒酶反轉錄酶(hTERT)。端粒酶的調控係由hTERT而非hTER的轉錄來決定。hTERT mRNA的即時聚合酶連鎖反應(PCR)因而是一種已獲認可的細胞端粒酶活性測定方法。 The function of telomerase is to synthesize telomere repeats, which are used to protect the integrity of chromosomes and extend the replication life of cells (Liu, K, et al., PNAS, 1999; 96: 5147-5152) . Telomerase is composed of two components, namely telomerase RNA template (hTER) and telomerase reverse transcriptase (hTERT). The regulation of telomerase is determined by hTERT rather than hTER transcription. The real-time polymerase chain reaction (PCR) of hTERT mRNA is therefore an approved method for measuring cell telomerase activity.
細胞分離:執行即時PCR實驗以測定人類臍帶組織衍生細胞的端粒酶生產。根據上文所述之實例製備人類臍帶組織衍生細胞。一般而言,得自國家疾病研究交換中心(Philadelphia,Pa.)的臍帶在正常分娩後會經過清洗以去除血液與碎屑然後進行機械解離。接著,將組織在37℃下以包括膠原蛋白酶、分散酶、及玻尿酸酶之消化酶於培養基中培養。根據上文實例中所述之方法培養人類臍帶組織衍生細胞。間葉幹細胞與正常皮膚纖維母細胞(cc-2509批號9F0844)係得自Cambrex,Walkersville,Md。多能性人類睪丸胚胎性癌(畸胎瘤)細胞系nTera-2細胞(NTERA-2 cl.Dl)(參見Plaia等 人,Stem Cells,2006;24(3):531-546)係購自ATCC(Manassas,Va.)並且將其根據上文中所述之方法來培養。 Cell isolation: Perform real-time PCR experiments to determine telomerase production of human umbilical cord tissue-derived cells. Human umbilical cord tissue-derived cells were prepared according to the examples described above. In general, umbilical cords obtained from the National Center for Disease Research and Exchange (Philadelphia, Pa.) Are washed after normal delivery to remove blood and debris and then mechanically dissociated. Next, the tissue was cultured in the culture medium at 37 ° C with digestive enzymes including collagenase, dispase, and hyaluronidase. Human umbilical cord tissue-derived cells were cultured according to the methods described in the examples above. Mesenchymal stem cells and normal skin fibroblasts (cc-2509 lot number 9F0844) were obtained from Cambrex, Walkersville, Md. The pluripotent human testicular embryonic carcinoma (teratoma) cell line nTera-2 cells (NTERA-2 cl. Dl) (see Plaia et al., Stem Cells, 2006; 24 (3): 531-546) were purchased from ATCC (Manassas, Va.) And cultivated according to the method described above.
總RNA分離:使用RNeasy®套組(Qiagen,Valencia,Ca.)將RNA自細胞萃取出來。用50微升經DEPC處理之水將RNA沖提出來然後儲存於-80℃下。使用隨機六聚物以TaqMan®反轉錄試劑(Applied Biosystems,Foster City,Ca.)來反轉錄RNA,反轉錄在25℃下歷時10分鐘、在37℃下歷時60分鐘、且在95℃下歷時10分鐘。將樣本儲存在-20℃下。 Total RNA isolation: RNA was extracted from cells using RNeasy® kit (Qiagen, Valencia, Ca.). RNA was extracted with 50 microliters of DEPC-treated water and stored at -80 ° C. Reverse transcription of RNA using TaqMan® reverse transcription reagent (Applied Biosystems, Foster City, Ca.) using random hexamers, reverse transcription at 25 ° C for 10 minutes, 37 ° C for 60 minutes, and 95 ° C for 95 minutes 10 minutes. Store samples at -20 ° C.
即時PCR:使用Applied Biosystems Assays-On-DemandTM(亦已知為TaqMan® Gene Expression Assays)根據製造商規範(Applied Biosystems)在cDNA樣本上執行PCR。此商用套組廣泛用來檢定人類細胞中的端粒酶。簡言之,使用7000序列偵測系統(搭載ABI prism 7000 SDS軟體(Applied Biosystems)),將hTert(人類端粒酶基因)(Hs00162669)及人類GAPDH(內部對照組)與cDNA及TaqMan® Universal PCR主混合物混合。熱循環條件初始為50℃歷時2分鐘然後95℃歷時10分鐘,接著進行40次95℃歷時15秒鐘然後60℃歷時1分鐘的循環。依據製造商規範來分析PCR數據。 Real-time PCR: Applied Biosystems Assays-On-Demand ™ (also known as TaqMan® Gene Expression Assays) was used to perform PCR on cDNA samples according to the manufacturer's specifications (Applied Biosystems). This commercial kit is widely used to detect telomerase in human cells. In short, using the 7000 sequence detection system (equipped with ABI prism 7000 SDS software (Applied Biosystems)), hTert (human telomerase gene) (Hs00162669) and human GAPDH (internal control group) and cDNA and TaqMan® Universal PCR The main mixture is mixed. The thermal cycle conditions were initially 50 ° C for 2 minutes and then 95 ° C for 10 minutes, followed by 40 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute. Analyze the PCR data according to the manufacturer's specifications.
人類臍帶組織衍生細胞(ATCC存取號PTA-6067)、纖維母細胞及間葉幹細胞係針對hTert及18S RNA來檢定。如表26-1中所示,hTert及因此端粒酶皆未在人類臍帶組織衍生細胞中偵測到。 Human umbilical cord tissue-derived cells (ATCC accession number PTA-6067), fibroblasts and mesenchymal stem cell lines were tested against hTert and 18S RNA. As shown in Table 26-1, neither hTert nor telomerase was detected in human umbilical cord tissue-derived cells.
人類臍帶組織衍生細胞(分離物022803,ATCC存取號PTA-6067)與nTera-2細胞皆經檢定,而結果顯示在兩批類臍帶組織衍生細胞中皆沒有端粒酶表現,然而畸胎瘤細胞系則顯示有高度表現(表26-2)。 Both human umbilical cord tissue-derived cells (isolate 022803, ATCC accession number PTA-6067) and nTera-2 cells were tested, and the results showed that neither batch of umbilical cord tissue-derived cells showed telomerase expression, but teratomas The cell line showed high performance (Table 26-2).
因此,可做出本發明之人類臍組織衍生細胞不表現端粒酶之結論。 Therefore, it can be concluded that the human umbilical tissue-derived cells of the present invention do not express telomerase.
本說明書中提及各種專利及其他出版物。這些出版物之各者之全部內容以引用方式併入本文中。 Various patents and other publications are mentioned in this specification. The entire contents of each of these publications are incorporated herein by reference.
儘管上文已藉由實例與較佳實施例說明本發明之各種態樣,但是應理解,本發明之範疇並不由前文描述限定,而是以下根據專利法原理正確解讀的申請專利範圍來限定。 Although the various aspects of the present invention have been described above by examples and preferred embodiments, it should be understood that the scope of the present invention is not limited by the foregoing description, but is limited by the following patent application scope correctly interpreted based on the principles of patent law.
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