TWI285219B - Method and composition for inducing neural differentiation - Google Patents
Method and composition for inducing neural differentiation Download PDFInfo
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- TWI285219B TWI285219B TW094120901A TW94120901A TWI285219B TW I285219 B TWI285219 B TW I285219B TW 094120901 A TW094120901 A TW 094120901A TW 94120901 A TW94120901 A TW 94120901A TW I285219 B TWI285219 B TW I285219B
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Description
1285219 ·
4 I 九、發明說明： 【發明所屬之技術領域】 本發明主要係關於一種無毒性誘發神經分化之方法及 組合物。 【先前技術】 中樞神經系統（CNS)損傷後的神經細胞喪失使得 CNS修復十分困難。許多研究顯示，分離自各種嚅齒類及 人類CNS區域之神經幹細胞（NSCs )在環境及/或外源 生長因子的影響下，可在成年嚅齒類CNS中分化為神經細 _ 胞（F· H· Gage, Mammalian neural stem cells· iSWe沉e 7 (2000) i岑33-7438 ; J· Price 及 Β·Ρ· Williams，Neural stem cells· Cw/r Opin. Neurobiol 11 (2001) 564-567 ) 〇 因此，補充 NSCs 被認 • 為是人類CNS治療的潛在策略（D· A· Peterson，Stem cells in brain plasticity and repair. Cum Opin. Pharmacol 2 (2002) 3442 ) 〇 衍生自人類骨髓之幹細胞（hBMSCs )在形態學上屬 於異源性。它們複潛在性地分化為骨母細胞、脂肪細胞、 • 軟骨細胞及肌肉，且亦可產生神經細胞（E. Mezey，K. J· Chandross，G. Harta，R. A. Maki 及 S. R. McKercher，Turning Blood into Brain: Cells Bearing Neuronal Antigens Generated in vivo from Bone Marrow. Science 290 (2000) 1779-1782 ; E· Mezey，S· Key， G· Vogelsang，I· Szalayova，G· D. Lange 及 B. Crain，Transplanted bone marrow generates new neurons in human brains, Proc. Natl Acad Scl U.SA. 700(2003) 1364-1369 ; Sanchez-Ramos 等人 1285219 ·
4 I (2000) ; D· Woodbury，Ε· J· Schwarz，D. J· Prockop 及 Ι· Β·
Black, Adult rat and human bone marrow stromal cells differentiate into neurons. 1 Neurosci. Res. 61 (2000) 364-370 )。近來已有報 導指出，人類及小鼠的BMSCs經視網酸及表皮生長因子 (EGF )或衍生自大腦之神經營養因子（BDNF )刺激 後’會表現神經祖細、胞標記物（巢蛋白（nestin ))、神 經細胞專一性核蛋白（NeuN )及神經膠纖維酸性蛋白 (GFAP ) ( Sanchez-Ramos 等人（2000))。亦已證明移植 的BMSCs可在損傷之CNS中分化為神經或神經膠世系（J· ^ R. Sanchez-Ramos, Neural cells derived from adult bone marrow and umbilical cord blood. J. Neurosci Res. 69 (2002) 880-893 )。在匕 外，Chopp等人發現移植BMSCs可促進患有局竈性腦絕血 - 之大鼠（J· Chen, Y· Li 及 M· Chopp，Intracerebral transplantation of bone marrow with BDNF after MCAo in rat. Neuropharmacology 39 (2000) 777-776 )、患有外傷性腦損傷之大鼠（〇丄11，丫· Li, L· Wang, J. Chen，A· Mahmood 及 Μ· Chopp，Intraarterial administration of marrow stromal cells in a rat model of traumatic • brain injury· J· 7S (2001) $/3-879 )及患有帕金森氏 症之小鼠（Υ· Li，J· Chen, L. Wang，L· Zhang，Μ· Lu 及 Μ· Chopp， Intracerebral transplantation of bone marrow stromal cells in a l-methyl-4-phenyl-l?2,3?6-tetrahydropyridine mouse model of Parkinson^ disease. Neurosci. Lett 316 (2001) 67-70 ) ^ ^ 13 41. ° 該等研究結果指出作為人類CNS細胞治療之有用細胞來源 的潛在角色。 1285219
然而，藉由培養平盤附著方式分離hBMSCs產生的主 要是異源族群（A· J· Friedenstein，J· E· Gorskaja 及 Ν· N· Kulagina, Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp. Hematol 4 (1976) 267-274 )。因此’ 已基於其相異尺寸及特定表面標記物而發展出數種方法’ 使用細胞分類產生同源族群（S. C. Hung，N. J· Chen，S· L· Hsieh，H. Li，H. L· Ma 及 W· H. Lo, Isolation and characterization of size-sieved stem cells from human bone marrow. Stem Cells 20 (2002) 2岑9-2兄；R· Zohar，J_ Sodek 及 C· A· McCulloch， Characterization of stromal progenitor cells enriched by flow cytometry. 90 (1997) )。據此，最近Hung 等人 ( 2002 )已發展出一種由人類骨髓有效分離同源族群之方 法，其係基於細胞尺寸及附著能力，使用Percoll梯度分離 及3-μιη孔篩去除較小的細胞。經純化而產生的hBMSC族群 稱為尺寸篩選細胞（SSCs)，其較hBMSC異源族群具有更 強的更新能力（Hung等人（2002)) aSSCs在轉種2至3缺 乏早期造血幹細胞的表面標記物CD34及AC133，且; 現成骨MSCs及成熟成骨前驅體之標記物（Hung等人 (2002))。然而，該等細胞表現Thy-Ι 、基質受體 (CD44 及 CD105 )及整合素（CD29 及 CD51 )。ssc
為複潛在性，在環境信號的影響下可生成成骨、成^旨_1 成軟骨世系（Hung等人（2002))。亦已發現以石_琉基乙 醇及視姻酸等抗氧化劑（其常用於在體外誘發幹細胞^神 經分化）刺激SSCs可電化產生神經細胞（s.C.Himg H 1285219 · < <
Cheng，C. Υ· Pan，Μ· J. Tsai, L· S· Kao 及 H. L. Ma，In vitro differentiation of size-sieved stem cells into electrically active neural cells· 20 (2002) 522-529 )。雖然 /3 -疏基乙醇及視 網酸對於SSCs分化為功能性神經細胞具有潛在效果，該二 因子在CNS組織修復的角色仍需界定。 然而，以/3 _巯基乙醇及視網酸等抗氧化劑刺激SSCs 而產生神經細胞僅能在體外施行。/3 ·髄基乙醇是一種有 毒試劑。此外，視網酸是一種致癌物。該二種抗氧化劑均 會對動物造成傷害，且移植經刺激的神經細胞會導致接受 I 者死亡。 【發明内容】 本發明提供一種將SSCs由纖維母細胞外形在形態上轉 變為具突起之外形的新穎方法，其係利用安全且可有效刺 ，激神經細胞形態變化之神經營養因子。此外，依據本發明 所得到之神經細胞適用於修復動物之神經缺損。 本發明之一目標係提供一種用於誘發神經分化之方 法，包括以神經.營養因子及/或二丁醯cAMP 齡 （dbcAMP )處理骨髓幹細胞，其中該神經營養因子包含 衍生自神經膠細胞株之神經營養因子（GDNF )或垂體腺 苷酸環化酶啓動肽（PACAP)。 本發明之另一目標係提供一種用於誘發神經分化之組 合物，包括神經營養因子及/或二丁醯cAMP ( dbcAMP)，其中該神經營養因子包含衍生自神經膠細 胞株之神經營養因子（GDNF )或垂體腺苷酸環化酶啓動 1285219 4 t 肽（PACAP) 〇 【實施方式】 本發明提供一種用於誘發神經分化之方法，包括以神 經營養因子及/或二丁醯cAMP ( dbcAMP )處理骨髓幹 細胞·，其中該神經營養因子包含衍生自神經膠細胞株之神 經營養因子（GDNF )或垂體腺苷酸環化酶啓動肽 (PACAP) 〇 本發明亦提供一種用於誘發神經分化之組合物，包括 神經營養因子及/或二丁醯cAMP ( dbcAMP )，其中該 > 神經營養因子包含衍生自神經膠細胞株之神經營養因子 (GDNF )或垂體腺苷酸環化酶啓動肽（PACAP)。 依據本發明，取用骨髓幹細胞以製備功能性神經細 胞。源自人類骨髓之幹細胞具有分化為神經細胞之潛力， 被認為是再生神經細胞的最佳材料。較佳者，該骨趨幹細 胞為源自人類骨髓之尺寸篩選幹細胞。尺寸篩選幹細胞的 開發係基於其相異尺寸及特定表面標記物以產生同源族 群，使用細胞分類以避免骨髓幹細胞初代培養物產生異源 | 族群。此外，尺寸篩選幹細胞較異源族群具有更強之更新 能力。較佳以3-μπι孔篩篩選源自人類骨髓之尺寸篩選幹細 胞。使用Percoll梯度分離及孔篩去除較小的細胞，即可基 於細胞尺寸及附著能力有效地由人類骨髓分離出尺寸篩選 幹細胞之同源族群。 依據本發明，以神經營養因子作為誘發神經分化之刺 激物。鑑於神經營養因子係存在於正常生理環境下之微環 1285219 « 1 境因子，較之化學試劑具有較小傷害性，其被認為是一種 處理用於移植至動物之骨髓幹細胞的安全試劑。依據本發 明，該神經營養因子包含衍生自神經膠細胞株之神經營養 因子或垂體腺苷酸環化酶啓動肽。 衍生自神經膠細胞株之神經營養因子（GDNF )對許 多CNS神經細胞族群而言係有力的存活因子，其對各種 CNS疾病具有治療潛力。最近Airaksinen及Saarma評論了 GDNF 的治療價值（M· S· Airaksinen及 M· Saarma，The GDNF family: signaling, biological functions and therapeutic value. Nat ^ Rev. Neuroscl 3 (2002) 383-394 )。亦有報告指出將 GDNF 以 脊椎内注射至受傷的脊髓，可促進脊髓損傷（SCI )大鼠 的後肢回復（H· Cheng, J· P. Wu 及 S· F· Tzeng，Neuroprotection of glial cell line-derived neurotrophic factor in damaged spinal cords following contusive injury. J. Neuroscl Res. 69 (2002) 397-405 ) 〇 依據本發明，GDNF處理可促進軸突及分支延伸突起的伸 展。在神經細胞專一性標記物方面，GDNF對神經纖維輕 蛋白質（NF-L )、液泡蛋白質一突觸素-1 ( synapsin-1 )及 _ 神經祖細胞標記物一中間黏連蛋白（internexin )的表現皆 具刺激作用。GDNF亦可在SSCs中誘發非專一性神經細胞 骨架蛋白質α -微管蛋白（α-tubulin )表現。較佳者，衍 生自神經膠細胞株之神經營養因子的劑量為2〇ng/mL至 50 ng/mL 〇 垂體腺苷酸環化酶啓動肽（PACAP )是一種誘 發之神經肽，其在活體内及活體外之CNS神經分化均扮演 1285219
• I 關鍵的角色（Ε· Dicicco-Bloom，Ν· Lu，J· Ε· Pintar 及 J. Zhang， The PACAP ligand/receptor system regulates cerebral cortical neurogenesis. Ann. ΝΎ. Acad Sci 11 (1998) 274-289 ； J. A.
Waschek，Multiple actions of pituitary adenylyl cyclase activating peptide in nervous system development and regeneration. Dev. Neurosci. 24 (2002) 14-23 )。此外，細胞内 cAMP 的提高對 軸突再生具決定性（W· D· Snider，F· Q· Zhou，J· Zhong 及 Α· Markus, Signaling the pathway to regeneration. Neuron 35 (2002) ) 。Gattei等人已發現GDNF受體-α及其附加酪 ® 胺酸激酶一 RET 在 hBMSCs 中表現（ν· Gattei，A. Celetti，Α·
Cerrato, Μ· Degan，A· De Iuliis，R Μ· Rossi，G· Chiappetta，C. Consales，S. Improta，V. Zagonel，D. Aldinucci，V· Agosti，M·
- Santoro, G. Vecchio, A· Pinto 及 M. Grieco, Expression of the RET receptor tyrosine kinase and GDNFR-alpha in normal and leukemic human hematopoietic cells and stromal cells of the bone marrow microenvironment. Blood 89 (1997) 2925-2937 )。依據未發明， PACAP係用於在SSCs形態上轉變為神經細胞時刺激神經細 馨胞分化。PACAP藉由與PAC1受體作用提高細胞内cAMP 而刺激神經生成（Dicicco_Bloom等人（1998))。依據本發 明，PACAP處理可促進軸突及分支延伸突起的伸展。在神 經細胞專一性標記物方面，PACAP對NF-L、液泡蛋白質一 突觸素-1及神經祖細胞標記物一中間黏連蛋白<的表現皆具 刺激作用。PACAP亦可在SSCs中誘發α -微管蛋白表現。 較佳者，垂體腺苦酸環化酶啓動肽的劑量為10 ng/mL至 1285219 20 ng/mL 〇
二丁醢cAMP〈 dbcAMP )是一種細胞可穿透性之 cAMP類似物，其可在SSCs中誘發高度分支、延長且纖細 之突起。依據本發明，dbcAMP處理可增加NF-L、液泡蛋 白質一突觸素-1及神經祖細胞標記物一中間黏連蛋白的表 現。dbcAMP亦可在SSCs中誘發α_微管蛋白表現。再 者，較之以GDNF及PACAP處理之SSC培養物，dbcAMP - 處理可造成更廣泛地分支及延長之突起。較佳者，二丁醯 cAMP的劑量為1〇〇 μΜ。 ® 在許多神經疾病中，神經營養因子對神經細胞存活及 修復具有效果。因此，細胞移植與神經營養因子的結合被 認為是神經疾病的有效治療策略。已證明神經營養因子可 - 誘發神經幹細胞分化（Ν· Υ· Ip，The neurotrophins and neuropoietic cytokines: two families of growth factors actingon neural and hematopoietic cells. Ann. KY. Acad. Sci 840 (1998) 97-106 ； A. Markus，T· D. Patel 及 W· D· Snider，Neurotrophic factors and axonal growth. Curr. Opin. Neurobioll2 (2002) 523-531 ； H. Thoenen, • Neurotrophins and neuronal plasticity. Science 270 (1995) 5幻-5卵）。然而神經營養因子對SSCs轉分化為神經細胞的 效果仍屬未知。本發明係首先顯示GDNF及PACAP可刺激 SSC朝向成熟神經細胞外型分化。已知這兩種神經營養因 子具神經保護效果，且可藉由活化cAMP/PKA、MAP激 酶、PI3激酶及PLC-γ訊號途徑而刺激軸突再生 (Airaksinen 等人（2002)及 Waschek 等人（2002))。此外， 1285219 « t 細胞内CAMP的提高可在SSCs中促進轴突形成。本發明顯 示培養於ITS培養基中之SSCs為具有突起之外形，且對神 經突觸液泡蛋白質一突觸素-1為陽性。在订8培養基中之 GDNF或PACAP處理可進一步誘發SsCs轉變為具纖細突起 之似神經細胞。已知表現於大多數CNS成熟神經細胞中之 NF蛋白質在神經細胞成長、組織、形狀及可塑性各方面 均扮演關鍵角色。因此’在SSCs中由含GDNF或PACAP之 ITS培養基所誘發之NF-L額外表現，表明該二種分子在 SSCs分化為神經細胞上的調控角色。在dbcAMp處理之 SSCs中所觀察到的延長突起廣泛分支以及仰丄水平增加， 暗示細胞内cAMP可能涉及促進PACAp處理之SSCs*化為 神經細胞。注意GDNF與PACAP或dbcAMP併用對SSCs中 NF-L及α -微管蛋白之產生並無加乘作用。 下列實例之目的僅供舉例說明，並無意限制本發明之 範_ 〇 實例1:源自人類骨婕之尺寸篩選幹細胞 SSCs係如先前所描述般分離自人類骨髓（Hung等人 > (2002))。簡言之，將人類骨髓由正常捐贈者之骼嵴吸 出，然後以磷酸鹽緩衝之生理鹽水（PBS )清洗二次。將 細胞放進1.073 g/ml Percoll溶液（Sigma® )，然後於900 g下 離心30分鐘。由交界面收集單核細胞（MNCs )，以1 X 106MNCs/cm2之密度置盤於1〇公分之塑膠培養皿，該培養 皿包含一插入之3-μηι孔篩（Transwell System，Coming® )。 將細胞培養於低匍萄糖之Dulbecco’s修改Eagle’s培養基 1285219 (DMFM-LG )中，該培養基包含ι〇〇/0胎牛血清 (FBS )、l〇〇U/ml 盤尼西林、l〇〇mg/ml鏈黴素及〇25 pg/ml兩性黴素B ( amphotericinB)(含血清培養笑）。 七天後，附著於插入篩上半部之細胞具有較大之似纖維母 細胞外形，將之命名為SSCs。然而，通過篩的細胞係呈小 多角形，且較少更新。接著以0.25%胰蛋白酶及imM EDTA收取SSCs，並重新置盤於1〇公分之培養皿上。當生 長於含血清培養基之SSCs達到80%密度時，將細胞以j χ 105細胞/皿之密度重新置盤於35毫米之培養皿上俾供西 方墨點分析，或以1 X 1〇4細胞/井之密度置於8井室中 俾供免疫螢光分析。 實例2 ··刺激源自人類骨髓之尺寸篩選幹細胞 於含血清培養基中經48小時後，以單獨之血清培養基 (ITS 培養基）及含 GDNF (20及 50ng/ml ; R&D® )、 PACAP ( 10 及 20 ng/ml ; Sigma®)或 dbcAMP ( 20 μΜ ； Sigma®)之ITS培養基處理SSCs。ITS培養基係由56% DMEM-LG ( Life Biotech⑧）、40% MCDB-201 培養基 (Sigma⑧）及含1 mg/ml·胰島素、0.55 mg/ml人類轉鐵蛋白 (transferrin )、0·5 pg/ml 亞硒酸鈉、10 nM 地塞米松 (dexamethasone ; Sigma® )及 10 μΜ 抗壞血酸（Sigma® ) 之1XITS培養基補充物（Sigma®)所組成。吾等發現不含 血清之DMEM-LG培養基中之SSCs對GDNF及PACAP反應 不佳。然而，當SSCs培養於具ITS補充物之不含血清培養 基時，該等細胞良好地附著於培養皿上並伸展其突起。因 ⑧ 12 1285219 此，於ITS培養基中進行處理。 SSCs之外形示於圖1及2。如圖2所示，當SSCs培養 於含10% FBS之培養基中時，該等細胞具有扁平而類似纖 維母細胞之外形。然而，發現當SSCs培養於單獨之ITS培 養基時會產生伸展之軸突（圖1及2)。此外，以含 GDNF或PACAP之ITS培養基處理SSCs進一步促進了軸突 的伸展。SSCs於單獨之ITS培養基培養7天後即可觀察到 液泡蛋白質突觸素-1。綜上所述，單獨之ITS培養基亦可 誘發SSCs在某種程度上分化為神經細胞。 ’實例3 : GDNF、PACAP及dbcAMP對神經細胞專一性標 記物之效果 本研究係關於GDNF、PACAP及dbcAMP對ITS培養 基中之SSCs分化為神經細胞的效果。以西方墨點法檢驗 SSCs中神經細胞專一性標記物（NF丄及NF-H )之水平。 將細胞以每30毫米培養孤1 x 1〇5個細胞之密度重新置 盤’並於含不同濃度GDNF、PACAP或dbcAMP之ITS培 養基中培養7天。收取細胞並使用含i〇/〇SDS 、ImM笨 基甲基磺基氟化物（PMSF )、lmMEDTA、h5mM胃蛋 白轉抑制劑（pepstatin )、2 mM leupeptin 及 0·7 mM 抑肽酶 (aprotinin )之PBS於冰上輕微同質化。使用Bio_Rad⑧ DC套組測定蛋白質濃度。將1〇叫總蛋白質裝載至 7.5%SDS-PAGE並轉移至硝基纖維素膜上。於4QC下將該 膜浸泡於抗NF抗體（Chemicon® )中過夜，接著浸泡於
辣根過氧化酶共軛二級抗體及化學冷光增強溶液（NEN ⑧ 13 1285219
LifeScience®)中，即可辨識NF_l ( 70kDa)及 Nr-Η (200kDa )。 結果示於圖3及4。參考圖3，西方墨點分析顯示以 GDNF或PACAP處理對SSCs中之NF-L蛋白質表現具有刺激 效果，雖然對SSCs中之NF-H水平效果較差。 如圖4所示，與在單獨之ITS培養基所觀察到者相 較，含細胞可穿透性cAMP類似物dbcAMP之ITS培養基 在SSCs中誘發了高度分支、延長且纖細之突起。如同 GDNF及PACAP ’西方墨點分析亦顯示北cAjy[p處理增加 了 SSCs中之NF-L及α ·微管蛋白水平。注意GDNF與 PACAP或dbcAMP併用對SSCs中NF_L及α -微管蛋白之產 生並無加乘作用。 對另一神經細胞中間絲蛋白質一 α -中間黏連蛋白之 免疫螢光染色（圖5 )係使用抗α -中間黏連蛋白抗體 (1 : 200 ; Chemicon® )進行。證據顯示 GDNF 及 PACAP在某個程度上可誘發SSCs之突起分支。然而，相較 於在以GDNF及PACAP處理之SSC培養物及對照組培養物 _ 所觀察到者，以dbcAMP處理7天導致突起更廣泛地分支 及延長。 實例4 : GDNF及PACAP對SSCs外形改變之效果 為了檢測經50 ng/ml GDNF或20 nM PACAP於ITS培養 基中處理7天後SSCs之外形改變，將SSCs於含PBS之4% 多聚甲醛中固定10分鐘。進行對突觸素-1——種突觸液泡 蛋白質之免疫螢光檢驗，係將SSCs與抗突觸素-1抗體 ⑧ 14
1285219 (1 : 200 ; BDBiosciences⑧）於 40C 下浸泡於含 〇 ι〇/ TritonX-100及5%馬血请之PBS中過夜，接著浸泡於生物 素化二級抗體（1 ·· 200 ; Vector®)及FITC-親和素 (1 : 200 ; Vector®)中。結果指出，在有處理或未處理 SSCs之突起及/·或周邊膜觀察到對突觸素-1的強烈免疫鸯 光染色。然而，證據指出在以GDNF及PACAP處理之拉養 物中，突觸素_1陽性細胞顯示伸展之具分支突起（如圖6 所示）。 雖然本發明之各種實施態樣已經例示及說明，熟習▲女 項技術者可進行各種修改及改良。希望本發明並不限 例示的特定形式，所有未偏離本發明精神及範疇之修改均 應涵括於下附申請專利範圍所定義的範疇内。 【圖式簡單說明】 圖1顯示SSCs似神經細胞轉形之相位差影像。其指出 在單獨之ITS培養基（〇)中以及在含5〇ng/mlGDNF或 20ng/mlPACAP之ITS培養基中，SSCs成為具突起之細胞 (放大倍率=200X )。 圖2顯示SSCs藉由GDNF、PACAP及dbcAMP之似神 經細胞轉形之相位差影像。其指出含血清培養基中之SSCs 外形類似纖維母細胞，並在撤回血清之培養基（單獨之 ITS培養基；0 )中成為具突起之形式。再者，於ITS培 養基中以 GDNF ( 50ng/ml )、PACAP ( 20ng/ml )、 dbcAMP (0‘lmM)、GDNF ( 50 ng/ml ) + PACAP (20 ng/ml )或 GDNF ( 50 ng/ml ) + dbcAMP ( 〇.i ⑧ 15 1285219 mM )處理7天之SSCs的突起為延長且高度分支者（放大 倍率=200 X )。 圖3顯示神經細胞專一性標記物NF-L及NF-H表現之 結果，其中西方墨點分析顯示相較於在單獨之ITS培養基 (0 )中所觀察到者，於ITS培養基中以GDNF ( 50 ng/ml )或 PACAP (10及 20ng/ml )處理 7 天之SSCs 中神 經細胞專一性標記物NF-L係經向上調節。 • 圖4顯示NF-L及α -微管蛋白表現之結果，其中西方 墨點分析顯示在如上述處理7天後之SSCs中，神經細胞專 ^ 一性標記物NF-L係經向上調節。此外，經由上述各種處 理，SSCs中之神經細胞專一性細胞骨架蛋白質α -微管蛋 白有所增加。單獨之ITS培養基處理以0表示。 “ 圖5顯示SSCs中α -中間黏連蛋白之免疫螢光染色結
_ 果，其中SSCs係在單獨之ITS培養基（0 )中或以GDNF (50 ng/ml )、PACAP ( 20 ng/ml )及 dbcAMP ( 0.1 mM )處理7天。然後對培養物進行α -中間黏連蛋白之 免疫螢光染色。以GDNF及dbcAMP處理之SSCs，其突起 • 之分支（箭號）及延長（箭頭）較單獨之ITS培養基 (0 )更為廣泛。比例尺=50 μηι。 圖6顯示SSCs似神經細胞轉形之影像。其指出當SSCs 培養於單獨之ITS培養基（0 )中7天時已經表現出液泡 蛋白質突觸素-1。此外，於ITS培養基中以GDNF ( 50 ng/ml )或PACAP ( 20ng/ml )處理7天可誘發進一步延 長（箭頭）及增加突起的分支（箭號）。比例尺=50 ⑧ 16 1285219
Claims (1)
- ,1285219 %年7月ή日修(更)正本 中華民國專利申請案第0941209011^~~^ 請範圍替換木〔2007丰5月） 十、申請專利範圍·· I 一種誘發神經分化之方法，包含以神經營養因子處 理骨髓幹細胞，其中該神經營養因子為衍生自神經 膠細胞株之神經營養因子（GDNF)或垂體腺苷酸環 化酶啓動肽(PACAP)。2·根據申請專利範圍第1項之方法，其中該骨髓幹細 胞為源自人類骨髓之尺寸篩選幹細胞。 3·根據申請專利範圍第2項之方法，其中該源自人類 骨髓之尺寸篩選幹細胞係以3-# m孔篩選。 4·根據申請專利範圍第丨項之方法，其中衍生自神經 膠細胞株之神經營養因子之含量為2〇ng/mL至 50ng/mL。 5·根據申請專利範圍第丨項之方法，其中垂體腺苷酸 環化酶啓動肽之含量為10ng/mL至2〇ng/mL。 6.根據申請專利範圍第丨項之方法，其中該神經分化 包括神經纖維輕蛋白質（NF-L)增加，α-微管蛋白 (α-tubulin)增加，液泡蛋白質一突觸素 -l(synapsin-l)產生，神經祖細胞標記物一中間黏蛋 白（internexin)產生，細胞突起延長，以及突起分支 增加。 7· —種用於處理骨髓幹細胞以誘發神經分化之組合 物，包含衍生自神經膠細胞株之神經營養因子 (GDNF)或垂體腺苷酸環化酶啓動肽(PACAP)。 8. 根據申請專利範圍第7項之組合物，其中該骨髓幹 細胞為源自人類骨髓之尺寸篩選幹細胞。 9. 根據申請專利範圍第7項之組合物，其中該源自人 類骨髓之尺寸篩選幹細胞係以3-//m孔篩選。 10. 根據申請專利範圍第7項之組合物，其中衍生 自神經膠細胞株之神經營養因子之含量為 20ng/mL 至 50ng/mL 0 11. 根據申請專利範圍第7項之組合物，其中垂體 腺苷酸環化酶啓動肽之含量為10ng/mL至 20ng/mL 0 12. 根據申請專利範圍第7項之組合物，其中該神 經分化包含神經纖維輕蛋白質（NF-L)增加，α-微 管蛋白（α-tubulin)增加，液泡蛋白質一突觸素 -l(synapsin-l)產生，神經祖細胞標記物一中間黏蛋 白（internexin)產生，細胞突起延長，以及突起分支 增加。
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| US8318485B2 (en) * | 2008-02-25 | 2012-11-27 | Natalie Gavrilova | Stem cell therapy for the treatment of diabetic retinopathy and diabetic optic neuropathy |
| US8333962B2 (en) * | 2009-08-27 | 2012-12-18 | National Health Research Institutes | Controlled release multidrug formulations for spinal cord injury |
| WO2012156968A2 (en) * | 2011-05-19 | 2012-11-22 | Ariel - University Research And Development Company, Ltd. | Use of mesenchymal stem cells for the improvement of affective and cognitive function |
| EP2968338B1 (en) * | 2013-03-15 | 2019-01-09 | Intra-Cellular Therapies, Inc. | Pde1 inhibitors for use in the treatment and/or prevention of cns injuries, and pns diseases, disorders or injuries |
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| US20030003090A1 (en) * | 2001-05-31 | 2003-01-02 | Prockop Darwin J. | Directed in vitro differentiation of marrow stromal cells into neural cell progenitors |
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