CN113454206A - 滑膜来源间充质干细胞及其用途 - Google Patents
滑膜来源间充质干细胞及其用途 Download PDFInfo
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- CN113454206A CN113454206A CN201980078739.5A CN201980078739A CN113454206A CN 113454206 A CN113454206 A CN 113454206A CN 201980078739 A CN201980078739 A CN 201980078739A CN 113454206 A CN113454206 A CN 113454206A
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- mesenchymal stem
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- derived mesenchymal
- hydrogel
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Abstract
本发明涉及使用滑膜组织和水凝胶来制备滑膜来源间充质干细胞的方法、通过该方法制备的滑膜来源间充质干细胞、包含该滑膜来源间充质干细胞的用于治疗骨或软骨损伤的药物组合物、包含滑膜组织和水凝胶的用于培养间充质干细胞的组合物以及试剂盒。通过使用本发明的制造方法能够有效地从滑膜中提取获得干细胞,且由此获得的滑膜来源间充质干细胞能够有效地治疗骨或软骨损伤,因此能够对治疗骨或软骨损伤的方法做出很大贡献。
Description
技术领域
本发明涉及滑膜来源间充质干细胞及其用途,更具体地,本发明涉及使用滑膜组织和水凝胶来制备滑膜来源间充质干细胞的方法、通过该方法制备的滑膜来源间充质干细胞、包含该滑膜来源间充质干细胞的用于治疗骨或软骨损伤的药物组合物、包含滑膜组织和水凝胶的用于培养间充质干细胞的组合物以及试剂盒。
背景技术
骨(bone)支撑着人体的软组织和体重,且围绕内部器官以保护内部器官免受外部冲击。此外,骨是人体的重要组成部分之一,不仅在结构上支撑肌肉和器官,而且储存体内的诸如钙和其他必需矿物质(即磷和镁)等物质。
在构成人体的骨之间存在关节。关节分为如头骨或牙根那样在彼此接触的两块骨或软骨之间几乎或完全没有活动性的不动关节、如动物的臂或腿部骨或颌骨那样在两块骨之间具有大量结缔组织且活动性大的可动关节、联合关节以及微动关节。通常,关节是指可动关节,该可动关节分为仅通过韧带连结两块骨的韧带连结和滑膜性连结。滑膜性连结是关节由结缔组织性的袋(关节囊)包裹而成的,其中关节囊内侧分泌起润滑剂作用的滑液,并且其外侧附着有许多韧带以增强关节。
干细胞(stem cell)是指处于分化为每个构成组织的细胞之前阶段的细胞,其能够在未分化状态下进行无限增殖,且具有通过特异性分化刺激分化为多种组织细胞的潜能。
干细胞根据其分化潜能大致分为胚胎干细胞(embryonic stem cell:ES cell)和成体干细胞(adult stem cell(组织特异性干细胞(tissue specific stem cell)))。胚胎干细胞是从在囊胚期(blastocyst)胚胎中待发育成胎儿的内细胞团(inner cell mass:ICM)中分离出来的干细胞,其具有能够分化为所有组织的细胞的潜力,其中囊胚期是受精卵形成后植入子宫内膜之前的早期阶段。
相反,组织特异性干细胞是在通过胚胎发育过程形成胚胎的每个器官的阶段中出现的每个器官特有的干细胞,其分化能力通常仅限于(多能(mulipotent))构成该组织的细胞。代表性的组织特异性干细胞包括存在于骨髓(bone-marrow)中的造血干细胞(hematopoietic stem cell)和分化为除血细胞以外的结缔组织(connective tissue)细胞的间充质干细胞(mesenchymal stem cell)。造血干细胞分化为诸如红细胞和白细胞的各种血细胞,间充质干细胞分化为成骨细胞(osteoblast)、成软骨细胞(chondroblast)、脂肪细胞(adipocyte)和成肌细胞(myoblast)等。
近年来,在成功地从人类分离出胚胎干细胞之后,其临床应用受到越来越多的关注。作为干细胞的应用领域,最受瞩目的是作为用于细胞替代疗法的细胞来源的用途。
在间充质干细胞分化为软骨形成细胞(chondrogenic cell),进一步分化为软骨细胞(chondrocyto)时,即进行成软骨分化(chondrogenic differentiation)时,细胞因子和生长因子会参与。尽管确切的机制尚不清楚,但已知TGF-β(转化生长因子β)、IGF(胰岛素样生长因子)、BMP(骨形态发生蛋白)、FGF(成纤维细胞生长因子)等对分化为软骨细胞的过程起到很重要的作用。因此,已有关于研究间充质干细胞的报道(美国授权专利第6835377号),该研究旨在将干细胞的上述分化能力应用于受损关节组织的再生和抗炎等治疗。另外,作为可用于治疗受损软骨的细胞,已在研究将骨髓干细胞(Majumdar M.K.等人,J.Cell.Physiol.(细胞生理学杂志)185:98-106,2000)、脐带血(Gang E.J.等人,Biochem.Biophys.Res.Commun.(生物化学和生物物理学研究通讯)321:102-108,2004)和滑膜(Fickert S.等人,Osteoarthritis Cartilage(骨性关节炎和软骨)11:790-800,2003)用作除患者的自体软骨细胞以外的替代细胞资源。但是,目前在使用干细胞的骨疾病或抗炎治疗中没有显着效果,特别是尚未有关于使用软骨干细胞的骨疾病或抗炎治疗的报道。
发明内容
技术问题
本发明的发明人为了开发可用于治疗软骨损伤的新干细胞而进行了多项研究,结果证实了来源于滑膜组织的间充质干细胞能够有效治疗软骨损伤,从而完成了本发明。
技术方案
本发明的主要目的是提供使用滑膜组织和水凝胶来制备滑膜来源间充质干细胞的方法。
本发明的另一目的是提供通过所述方法制备的滑膜来源间充质干细胞。
本发明的又一目的是提供用于治疗骨或软骨损伤的药物组合物,其包含所述滑膜来源间充质干细胞。
本发明的又一目的是提供用于培养间充质干细胞的组合物,其包含滑膜组织和水凝胶。
本发明的又一目的是提供用于培养间充质干细胞的试剂盒,其包含所述组合物。
本发明的又一目的是提供滑膜来源间充质干细胞在制备用于治疗骨或软骨损伤的药物组合物中的用途。
发明效果
通过使用本发明的制造方法能够有效地从滑膜中提取获得干细胞,且由此获得的滑膜来源间充质干细胞能够有效地治疗骨或软骨损伤,因此能够对治疗骨或软骨损伤的方法做出很大贡献。
附图说明
图1a是示出将滑膜切片培养14天的结果的显微照片。
图1b是示出将synMSC传代培养15代的结果的显微照片,其中传代1(Passage 1)表示第1代(P1),传代5(Passage 5)表示第5代(P5),传代15(Passage 15)表示第15代(P15)。
图2a是示出用针对synMSC阳性表位的单克隆抗体进行免疫染色的结果的照片。
图2b是示出用针对synMSC的阴性表位的单克隆抗体进行免疫染色的结果的照片。
图2c是示出对在对synMSC进行免疫染色时显色的荧光进行定量分析的结果的图。
图3a是示出在诱导synMSC分化为脂肪细胞后进行油红O(Oil Red O)染色的结果的荧光显微照片。
图3b是示出对从分化自synMSC的脂肪细胞中测量到的油红O染色水平进行定量分析的结果的图。
图3c是示出在诱导synMSC分化为骨细胞后进行茜素红S(Alizarin Red S)染色的结果的荧光显微照片。
图3d是示出对从分化自synMSC的骨细胞中测量到的茜素红S染色水平进行定量分析的结果的图。
图3e是示出在诱导synMSC分化为骨细胞后由ALP活性引起的BCIP/NBT显色水平的荧光显微照片。
图3f是示出从分化自synMSC的骨细胞中测量到的ALP活性进行定量分析的结果的图。
图4a是示出对作为对照组的BMSCs和P3 synMSC进行H&E染色和阿尔辛蓝(alcianblue)染色的结果的显微照片。
图4b是示出对BMSCs和P3 synMSC的H&E染色水平进行定量分析的结果的图,图4c是示出对BMSCs和P3 synMSC的阿尔辛蓝染色水平进行定量分析的结果的图。
图5a是示出对从P3 synMSC(对照组)、分化自P3 synMSC的软骨细胞(比较组)以及使用BMP-7来分化自P3 synMSC的软骨细胞(实验组)中测量到的sGAGs(硫酸化糖胺聚糖)水平进行定量分析的结果的图。
图5b是示出对P3 synMSC(对照组)、分化自P3 synMSC的软骨细胞(比较组)以及使用BMP-7来分化自P3 synMSC的软骨细胞(实验组)所表达的软骨标志物蛋白(SOX-9,聚集蛋白聚糖(aggrecan)和Ⅱ型胶原蛋白)的表达水平进行测量的结果的电泳照片。
图5c是示出对P3 synMSC(对照组)、分化自P3 synMSC的软骨细胞(比较组)和使用BMP-7来分化自P3 synMSC的软骨细胞(实验组)所表达的软骨标志物蛋白(SOX-9,聚集蛋白聚糖(aggrecan)和Ⅱ型胶原蛋白)的表达水平进行定量分析的结果的图。
图6a是示出在使用PLGA支架培养P3 synMSC之后分析活细胞数量变化的结果的图。
图6b是示出在使用PLGA支架培养P3 synMSC时随培养时间的形态变化的扫描电子显微照片。
图7是示出对将PLGA支架(PLGA scaffold)、在PLGA支架上培养synMSC而得的培养物(PLGA/SynMSC)以及在载有BMP-7的PLGA支架上培养synMSC而得的培养物(PLGA/SynMSC/BMP-7)移植的兔关节的股骨进行H&E染色、番红O(Safranin-O)染色和利用Ⅱ型胶原蛋白抗体的免疫染色的结果的显微照片。
具体实施方式
为了实现上述目的的本发明的一实施方式提供了使用滑膜组织和水凝胶来制备滑膜来源间充质干细胞的方法以及通过该方法制备的滑膜来源间充质干细胞。具体地,制备滑膜来源间充质干细胞的方法包括:(a)使滑膜组织包埋在水凝胶中并进行培养以获得培养物;以及(b)通过在所获得的培养物中分解水凝胶来回收从所述滑膜组织迁移至水凝胶中并增殖的间充质干细胞。
本发明的术语“滑膜(synovium、synovial membrane、synovial stratum)”也称为润滑膜或滑液膜,是指构成围绕关节腔的关节囊的内层的塑性结缔组织,已知滑膜具有发达的毛细血管,从而活跃地进行滑液交换。滑膜根据关节的不同而具有不同的体积,在某些情况下呈现形成有皱纹以在关节腔中围绕脂肪体并填充关节腔的形态。
在本发明中,滑膜可以理解为用于分离间充质干细胞的来源组织。
本发明的术语“水凝胶”是指含有水作为分散介质的凝胶。水凝胶主要通过以下方式生成:由于冷却而丧失流动性,或具有三维网状结构和微晶结构的亲水性聚合物因含有水而溶胀。含有电解质聚合物的水凝胶表现出高吸水性,因此已在许多领域中作为吸水性聚合物得到实用化。
在本发明中,只要构成水凝胶的亲水性聚合物可用于滑膜来源间充质干细胞的制备过程,则对其没有特别限制,例如,可以是胶原蛋白(collagen)、明胶(gelatin)、软骨素(chondroitin)、透明质酸(hyaluronic acid)、藻酸(alginic acid)、基质胶(MatrigelTM)、壳聚糖(chitosan)、肽(peptide)、纤维蛋白(fibrin)、聚乙醇酸(PGA)、聚乳酸(PLA)、聚乙二醇(PEG)或聚丙烯酰胺(polyacrylamide),也可以是它们的混合物。
在本发明提供的制备滑膜来源间充质干细胞的方法的步骤(a)中,对使滑膜组织包埋在水凝胶中的方法没有特别限制,且可通过如下方法进行:将滑膜组织和构成水凝胶的亲水性聚合物混合并将该亲水性聚合物变换为水凝胶;或形成水凝胶并将滑膜组织以物理方式引入水凝胶内部。
在步骤(a)中,可以在将含有滑膜组织的水凝胶浸入本领域已知适于干细胞培养的常规培养基中之后,对含有滑膜组织的水凝胶进行培养。
对培养基没有特别限制,作为一例,可以使用DMEM(Dulbecco的改良Eagle培养基)或角质形成细胞-SFM(角质形成细胞无血清培养基),并且作为另一例,可以使用D-培养基(Gibco)。
培养基可以进一步包含多种类型的添加剂。例如,等渗溶液中的中性缓冲液(例如磷酸盐和/或高浓度碳酸氢盐)、蛋白质营养物质(例如血清(例如FBS)、血清替代物、白蛋白或必需氨基酸和非必需氨基酸(例如谷氨酰胺))、脂质(脂肪酸、胆固醇、血清的HDL或LDL提取物)、其他成分(例如胰岛素或转铁蛋白、核苷或核苷酸、丙酮酸盐、任一离子化形态或盐的糖源(例如葡萄糖)、硒、糖皮质激素(例如氢化可的松)和/或还原剂(例如β-巯基乙醇))等可用作为添加剂。另外,为了防止细胞粘接等,也可以将抗结团剂(anti-clumpingagent)用作添加剂。
另外,在本发明提供的制备滑膜来源间充质干细胞的方法的步骤(b)中,只要在不影响水凝胶中的干细胞的情况下能够分解水凝胶,则对水凝胶的分解没有特别限制,作为一例,可以通过利用酶反应的方法进行,作为另一例,可以通过使用可裂解形成水凝胶的亲水性聚合物的键的酶(诸如,胶原酶、明胶酶、尿激酶、链激酶、TPA(组织纤溶酶原激活剂)、纤溶酶(plasmin)、透明质酸酶等)的方法进行。
另一方面,通过本发明提供的制备滑膜来源间充质干细胞的方法所制备的间充质干细胞表现出在其细胞表面表达CD29、CD44、CD73和CD90的免疫学特性,并且表现出能够分化为选自由脂肪细胞、骨细胞、软骨细胞及其组合组成的组中的细胞的特性。
特别地,在将间充质干细胞分化为软骨细胞时,用BMP-7处理能够显着提高分化为软骨细胞的效率。
另外,PLGA支架的使用能够促进间充质干细胞的增殖。
本发明的另一实施方式提供了用于治疗骨或软骨损伤的药物组合物,其包含滑膜来源间充质干细胞、其培养物或从间充质干细胞分化的细胞作为活性成分。
本发明的术语“治疗”是指通过滑膜来源间充质干细胞、其培养物或从间充质干细胞分化的细胞的给药来使骨或软骨损伤的症状好转或得到改善的所有行为。
根据本发明的一实施方式,在将滑膜来源间充质干细胞注射到膝关节软骨和股骨的受损部位后,确认到表现出再生受损骨和软骨的治疗效果且BMP-7提升该治疗效果。
在本发明中,滑膜来源间充质干细胞能够表现出治疗受损的骨或软骨的效果,并且在使用将间充质干细胞与支架混合而得的形态时能够提升该治疗效果。具体地,在使用以滑膜来源间充质干细胞附着在支架上的形态培养而得的培养物时能够提升该治疗效果,并且在使用具有在表面上载有BMP-7的形态的支架时能够进一步提升该治疗效果。
在本发明中,只要支架不影响滑膜来源间充质干细胞的治疗效果,则对支架没有特别限制,例如,其可以是PLGA支架。
对本发明提供的药物组合物中包含滑膜来源间充质干细胞的程度没有特别限制,作为一例,每1ml中可包含1.0×105至1.0×109个细胞,作为另一例,每1ml中可包含1.0×106至1.0×108个细胞,作为又一例,每1ml中可包含1.0×107个细胞。
另外,药物组合物可以按照药学领域中的常规方法配制成适合患者体内给药的单位剂型制剂以进行给药,并且制剂可以包含根据单次或多次给药的有效剂量。适合此目的的剂型优选地作为肠胃外制剂包括注射剂(例如用于注射的安瓿)、输注剂(例如输液袋)以及喷雾剂(例如气雾剂)。用于注射的安瓿可以在即将使用前与注射液混合调配,并且可以将生理盐水、葡萄糖、甘露醇、林格氏液等用作注射液。另外,输液袋可以由聚氯乙烯或聚乙烯制成,并且可以例举Baxter、Becton Dickinson、Medcep、National Hospital Products或Terumo公司的输液袋。
在药物组合物中,除滑膜来源间充质干细胞外,可进一步包含一种或多种药学上可接受的常规惰性载体,例如,对于注射剂,可进一步包含防腐剂、无痛剂、增溶剂或稳定剂等,对于局部给药制剂,可进一步包含基剂、赋形剂、润滑剂或防腐剂等。
另外,在本发明提供的药物组合物中,除滑膜来源间充质干细胞外,可进一步包含辅助治疗骨或软骨损伤、维持滑膜来源间充质干细胞的活性、或促进滑膜来源间充质干细胞的分化的多种成分,例如抗炎剂、干细胞动员因子、生长诱导因子等。
本发明的药物组合物可通过本领域常用的给药方法与用于移植和其他用途的其他干细胞一同以混合物的形态进行给药,优选地可直接植入或移植至需要治疗的患者的患病部位上或直接移植至或注射至腹腔中,但不限于此。此外,给药可以采用使用导管的非手术给药方法,也可以采用诸如切开患病部位后进行注射或移植等的手术给药,但是更优选采用使用导管的非手术给药方法。另外,除了根据常规方法进行胃肠外给药(例如直接给药至病变部位)外,也可以通过血管内注射进行移植,这是造血干细胞移植的常规方法。
对干细胞的日剂量没有特别限制,作为一例,可以按1.0×104至1.0×1010个细胞/kg体重进行单次或分多次给药,作为另一例,可以按1.0×105至1.0×109个细胞/kg体重进行单次或分多次给药。但是,应当理解,活性成分的实际剂量应根据诸如要治疗的疾病、疾病的严重程度、给药途径、患者的体重、年龄和性别等多种相关因素来确定,因此上述剂量在任何方面都不限定本发明的范围。
本发明的又一实施方式提供包含滑膜组织和水凝胶的用于培养间充质干细胞的组合物以及包含该组合物的用于培养间充质干细胞的试剂盒。
如上所述,当将滑膜组织包埋在水凝胶中并进行培养时,能够有效地制备滑膜来源间充质干细胞,因此包含滑膜组织和水凝胶的组合物能够用作用于培养间充质干细胞的组合物。
另外,用于培养间充质干细胞的试剂盒可包括用于培养间充质干细胞的组合物以及诸如培养间充质干细胞所需的溶液和设备等多种组成部分。
对组成部分没有特别限制,例如可以是试管、适当的容器、反应缓冲液(pH和缓冲液浓度不同)、培养容器、用于培养的培养基、无菌水等。
本发明的又一实施方式提供了滑膜来源间充质干细胞在制备用于治疗骨或软骨损伤的药物组合物中的用途。
用于制备药物组合物的滑膜来源间充质干细胞可以与可接受的佐剂、稀释剂、载体等混合,并且可以与其他活化剂一起制成复合制剂以具有活性成分的增强作用。
在本发明的组合物、用途和治疗方法中提及的事项彼此同样地适用,除非彼此矛盾。
本发明实施例
在下文中,将通过实施例更详细地描述本发明。然而,以下实施例仅用于对本发明的示例性说明,并且本发明的内容不限于这些实施例。
实施例1:获得滑膜来源间充质干细胞
滑膜是从兔子(新西兰白兔子,6-48周大,雄性)的两个膝关节的髌上囊(suprapatellar pouch)获得的。将获得的滑膜切碎以获得滑膜切片,然后用PBS清洗。将清洗后的滑膜切片悬于含有100μg/ml氨基甲基苯甲酸且溶解有1单位(unit)/ml凝血酶的DMEM培养基中,并将该DMEM培养基与含有40mmol/L氯化钙且溶解有0.5%纤维蛋白原的DMEM培养基以1:1(v/v)混合以形成水凝胶。将包含约200mg滑膜切片的10ml水凝胶放入100mm培养容器中,并在37℃下在加湿室中培养2小时,然后对其添加10ml初级生长培养基(90%DMEM-Ham's F12、10%胎牛血清、10ng/ml表皮生长因子(EGF)、2ng/ml碱性成纤维细胞生长因子(bFGF)、10ng/ml胰岛素样生长因子(IGF)和10μg/ml庆大霉素),在37℃下在加湿室中培养14天。培养结束后,除去培养基,添加含有5000单位尿激酶和30%小牛血清的DMEM培养基,分解成纤维蛋白的水凝胶,然后进行离心分离(150g,5分钟),获得滑膜来源间充质干细胞(synMSC)(图1a)。
图1a是示出将滑膜切片培养14天的结果的显微照片。
用PBS将获得的synMSC清洗两次,并悬于二级生长培养基(含10%v/v FBS、10U/ml抗生素、10ng/ml EGF和2ng/ml bFGF的DMEM/Ham's F-12(1,1)混合物)中,然后传代培养7天,当达到80至90%的饱和度时,终止培养。然后,用2.5%(w/v)胰蛋白酶-EDTA溶液处理该培养物以分离细胞,然后进行离心分离,获得第1代(P1)synMSC。此后,重复执行相同的方法,获得五个不同系列的第15代(P15)synMSC(图1b)。
图1b是示出将synMSC传代培养15代的结果的显微照片,其中传代1(Passage 1)表示第1代(P1),传代5(Passage 5)表示第5代(P5),传代15(Passage 15)表示第15代(P15)。
如图1b所示,可确认滑膜来源间充质干细胞(synMSC)即使在传代培养15次后也能够保持其形态特征。
实施例2:synMSC的免疫表型分析
在实施例1中获得的各传代世代的synMSC中,对第三代(P3)的synMSC分析了细胞表面的表位谱(epitope profile)。此时,表位是CD29、CD34、CD44、CD45、CD73和CD90。
在96孔板中大约每孔分配1×104个P3 synMSC并进行培养,在培养结束后添加含1%BSA的PBST(含0.05%Tween 20的PBS)以进行封闭。随后,添加了针对上述表位的每种单克隆抗体(抗小鼠CD29、抗兔CD44、抗人CD73 PE-Cyanine7、抗人CD90 PE-CyTM7、抗CD34PerCPCy5.5和抗兔CD45)并使得进行反应。在反应结束后,用PBS将细胞清洗3次,添加1%BSA和二抗(用Alexa Fluor 488标记的山羊抗小鼠IgG,1:100),然后使得在暗室中进一步反应。在反应结束后,再次用PBS将细胞清洗3次,添加DAPI(分子探针,俄勒冈州,美国),然后在室温暗室中进行复染(counterstaining)。在染色结束后,用Operetta174;高内涵成像系统(PerkinElmer,马萨诸塞州,美国)扫描,对荧光强度进行定量分析(图2a至2c)。此时,作为对照组,使用Alexa Fluor 488山羊抗小鼠IgG(H+L)代替针对表位的单克隆抗体。
图2a是示出用针对synMSC阳性表位的单克隆抗体进行免疫染色的结果的照片,图2b是示出用针对synMSC的阴性表位的单克隆抗体进行免疫染色的结果的照片,图2c是示出对在对synMSC进行免疫染色时显色的荧光进行定量分析的结果的图。
如图2a至2c所示,证实了在synMSC的细胞表面上表达了CD29、CD44、CD73和CD90,并且未表达CD34和CD45。
实施例3:synMSC分化能力的分析
实施例3-1:分化为脂肪细胞
对装有脂肪分化诱导培养基(含0.5mM异丁基甲基黄嘌呤、1μM地塞米松、10μM胰岛素、200μM吲哚美辛、1%抗生素的DMEM)的48孔板每孔分配1×104个P3 synMSCs,以单层培养8天以诱导分化为脂肪细胞。作为对照组,使用在基础培养基(含10%FBS的DMEM)中培养而得的培养物。用4%多聚甲醛处理已培养结束的细胞并进行固定,并且在进行油红O染色以检测作为脂肪细胞特征的细胞内脂质液泡之后,用荧光显微镜进行观察(图3a)。此后,为了定量分析染料的含量,向染色的细胞中添加异丙醇以提取染料,并测量540nm处的吸光度(图3b)。
图3a是示出在诱导synMSC分化为脂肪细胞后进行油红O(Oil Red O)染色的结果的荧光显微照片,图3b是示出对从分化自synMSC的脂肪细胞中测量到的油红O染色水平进行定量分析的结果的图。
如图3a和3b所示,在诱导synMSC分化为脂肪细胞时,在8天后在细胞中检测到脂质液泡,这一点通过油红O染色得到验证。
因此,可知synMSC表现出分化为脂肪细胞的能力。
实施例3-2:分化为骨细胞
对含有骨形成诱导培养基(悬浮有100nM地塞米松、50μg/ml抗坏血酸-2-磷酸酯、10mM的β-甘油磷酸酯、1%抗生素的DMEM)的48孔板每孔分配1×104个P3 synMSC,培养14天以诱导分化为骨细胞。作为对照组,使用在基础培养基(含10%FBS的DMEM)中培养而得的培养物。用4%多聚甲醛处理培养结束的细胞并进行固定,并添加作为ALP(碱性磷酸酶)的底物的BCIP/NBT(Sigma Aldrich,密苏里州,美国)并使得进行反应。在反应结束后,进行茜素红S染色以检测作为骨细胞特征的钙质,然后用荧光显微镜进行观察,并对其染色水平进行定量分析(图3c和3d)。
图3c是示出在诱导synMSC分化为骨细胞后进行茜素红S染色的结果的荧光显微照片,图3d是示出对从分化自synMSC的骨细胞中测量到的茜素红S染色水平进行定量分析的结果的图。
如图3c和3d所示,在诱导synMSC分化为骨细胞时,在14天后在细胞中检测到钙化,这一点通过茜素红S染色得到证实。
另外,图3e是示出在诱导synMSC分化为骨细胞后由ALP活性引起的BCIP/NBT显色水平的荧光显微照片,图3f是示出从分化自synMSC的骨细胞中测量到的ALP活性进行定量分析的结果的图。
如图3e和3f所示,证实了在诱导synMSC分化为骨细胞时,已知为骨分化标志物的ALP的活性在14天后增加约3倍。
因此,可知synMSC表现出分化为骨细胞的能力。
实施例3-3:分化为软骨细胞
对装有软骨诱导培养基(悬浮有1%小牛血清、1×ITS、0.1mM地塞米松、50μg/ml抗坏血酸-2-磷酸的DMEM)的多聚赖氨酸包被6孔培养板每孔分配1×106个P3 synMSC并培养2周以诱导分化为软骨细胞。在培养的第二天,由P3 synMSC形成了球体,每两天更换一次培养基。在培养结束后,得到球体作为培养产物,用4%的多聚甲醛对其进行处理2小时并进行固定。然后,进行离心分离(300g,5分钟),得到球体,用PBS清洗3次后,依次用含量从50%增加至100%的乙醇溶液进行处理以使球体脱水。然后,将球体包埋在石蜡中,获得4μm厚的薄片,然后进行H&E染色和阿尔辛蓝染色。核部位用核固红进行复染(图4a至4c)。此时,作为对照组,使用在相同条件下诱导BMSCs(骨髓来源干细胞)分化的结果物。
图4a是示出对作为对照组的BMSCs和P3 synMSC进行H&E染色和阿尔辛蓝染色的结果的显微照片,图4b是示出对BMSCs和P3 synMSC的H&E染色水平进行定量分析的结果的图,图4c是示出对BMSCs和P3 synMSC的阿尔辛蓝染色水平进行定量分析的结果的图。
如图4a至4c所示,当在相同条件下进行软骨分化时,确认到从P3 synMSC分化出软骨细胞的水平显着高于从作为对照组的BMSC分化出软骨细胞的水平。特别是,通过H&E染色确认到,分化自P3 synMSC的软骨细胞与分化自BMSCs的软骨细胞相比呈现高约9倍的水平,并且通过阿尔辛蓝染色确认到,分化自P3 synMSC的软骨细胞与分化自BMSCs的软骨细胞相比呈现高约13倍的水平。
因此,可知synMSC表现出分化为软骨细胞的能力。
实施例3-4:BMP-7对软骨细胞分化的作用
实施例3-4-1:使用BMP-7诱导synMSC分化为软骨细胞
除了使用添加有BMP-7(50ng/ml)的软骨诱导培养基以外,以与实施例3-3相同的方式诱导P3 synMSC分化为软骨细胞。此时,作为对照组,采用了使用基础培养基(含10%FBS的DMEM)而非软骨诱导培养基的培养物,并且作为比较组,采用了以与实施例3-3相同的方式诱导P3 synMSC分化为软骨细胞而得的培养物。
实施例3-4-2:sGAGs(硫酸化糖胺聚糖)含量分析
定量分析实施例3-4-1中获得的对照组、比较组和实验组的SGAG(硫酸化糖胺聚糖)。
大致地将Blyscan染料添加到每个样品的细胞提取物中,在振荡条件下反应30分钟,然后进行离心分离(12000rpm,10分钟)以获得沉淀物。将解离试剂(dissociationreagent)添加到获得的沉淀物中以获得悬浮液,并在656nm处测量吸光度(图5a)。
图5a是示出对从P3 synMSC(对照组)、分化自P3 synMSC的软骨细胞(比较组)以及使用BMP-7来分化自P3 synMSC的软骨细胞(实验组)中测量到的sGAGs(硫酸化糖胺聚糖)水平进行定量分析的结果的图。
如图5a所示,确认到与分化自P3 synMSC的软骨细胞(比较组)相比,使用BMP-7来分化自P3 synMSC的软骨细胞(实验组)中检测到的sGAGs水平明显更高。
实施例3-4-3:软骨标志物表达水平的分析
使用RT-PCR分析了实施例3-4-1中获得的对照组、比较组和实验组的软骨标志物蛋白(SOX-9,聚集蛋白聚糖(aggrecan)和Ⅱ型胶原蛋白)的表达水平。
大致地,破坏每个样品的细胞,并使用Trizol试剂(Invitrogen,加利福尼亚州)提取各自的总RNA。将每个提取的总RNA施加于TOPscriptTM一步式RT PCR试剂盒(Enzynomics,大田,韩国)以合成每个cDNA。将每个合成的cDNA用作模板,并使用以下引物进行PCR以获得各自的扩增产物,并对其水平进行定量分析(图5b和5c)。此时,GAPDH被用作内部对照组。
SOX-9F:5'-CCCGATCTGAAGAAGGAGAGC-3'(序列号1)
SOX-9R:5'-GTTCTTCACCGACTTCCTCCG-3'(序列号2)
聚集蛋白聚糖F:5'-TGAGGAGGGCTGGAACAAGTACC-3'(序列号3)
聚集蛋白聚糖R:5'-GGAGGTGGTAATTGCAGGGAACA-3'(序列号4)
T2胶原蛋白F:5'-TTCAGCTATGGAGATGACAATC-3'(序列号5)
T2胶原蛋白R:5'-AGAGTCCTAGAGTGACTGAG-3'(序列号6)
GAPDH F:5'-ATTGTTGCCATCAATGACCC-3'(序列号7)
GAPDH R:5'-AGTAGAGGCAGGGATGATGTT-3'(序列号8)
图5b是示出对P3 synMSC(对照组)、分化自P3 synMSC的软骨细胞(比较组)以及使用BMP-7来分化自P3 synMSC的软骨细胞(实验组)所表达的软骨标志物蛋白(SOX-9,聚集蛋白聚糖(aggrecan)和Ⅱ型胶原蛋白)的表达水平进行测量的结果的电泳照片。图5c是示出对P3 synMSC(对照组)、分化自P3 synMSC的软骨细胞(比较组)和使用BMP-7来分化自P3synMSC的软骨细胞(实验组)所表达的软骨标志物蛋白(SOX-9,聚集蛋白聚糖(aggrecan)和Ⅱ型胶原蛋白)的表达水平进行定量分析的结果的图。
如图5b和5c所示,确认到与分化自P3 synMSC的软骨细胞(比较组)相比,使用BMP-7来分化自P3 synMSC的软骨细胞(实验组)的软骨标志物蛋白(SOX-9,聚集蛋白聚糖和Ⅱ型胶原蛋白)表达水平显着更高。
实施例4:PLGA支架对细胞增殖的影响
实施例4-1:PLGA支架的制备
将通过将PLGA溶解在二氯甲烷(DCM)中而获得的20%(w/v)的聚合物溶液装在配备有17号针头的10ml注射器中并进行湿纺,然后使用辊获得PLGA纤维。将获得的PLGA纤维干燥48小时,然后在-70℃下真空干燥3天以去除残留的溶剂。
使用获得的PLGA纤维来形成三维PLGA支架,将其浸入1.0M PBS(pH 7.4)中,并在37℃下以60rpm搅拌,以除去由PLGA降解的酸性副产物。用PBS清洗除去了酸副产物的PLGA支架3次,并真空干燥48小时,以制备用于培养P3synMSC的三维支架。
实施例4-2:PLGA支架的效果
用乙醇处理在实施例4-1中制备的PLGA支架以进行润湿后,每片接种1×106个P3synMSC,用PBS清洗两次,然后在二级生长培养基中培养7天。在培养结束后,使用比色CCK-8测定法(Dojindo Molecular Technologies Inc.,马里兰州,美国)分析活细胞数,并用扫描电子显微镜(SEM)分析形态(图6a和6b)。此时,作为对照组,使用了在相同条件下在24孔板中培养P3 synMSC而得的培养物。
图6a是示出在使用PLGA支架来培养P3 synMSC之后分析活细胞数变化的结果的图,图6b是示出在使用PLGA支架来培养P3 synMSC时随培养时间的形态变化的扫描电子显微照片。
如图6a和6b所示,在使用PLGA支架而不是24孔板时,确认到P3 synMSC的培养效率提高,而据分析,这是因为P3 synMSC对PLGA支架的粘附率随时间增加且由此引起增殖率的提高。
实施例4-3:载有BMP-7的PLGA支架的制备
将重组人BMP-7蛋白(3μg)溶解在10μL的PBS中,然后在丙酮/乙醇(9:1)溶剂中与0.2%(w/v)PLGA一同进行乳化,由此获得BMP-7的油包水(water-in-oil)悬浮液(1:100w/o的比例),将其装在配有17号针头的10ml注射器中,并通过电喷雾法(10kV电压,0.033ml/分钟的流速)在PLGA纤维上进行纺丝,从而制备具有载有封装在PLGA纤维上的BMP-7的形态的PLGA支架。
实施例5:使用synMSC治疗软骨损伤
分别准备实施例4-1中制备的PLGA支架、实施例4-2中制备的在PLGA支架上培养synMSC而得的培养物(PLGA/SynMSC)以及在实施例4-3中制备的载有BMP-7的PLGA支架上培养synMSC而得的培养物(PLGA/SynMSC/BMP-7),并使用它们制备了分别具有1mm厚度和10mm长度的植入物。
同时,将甲苯噻嗪(5mg/kg)肌肉注射到平均体重为3.2kg的雄性新西兰白兔中,随后进行全身麻醉,并在膝关节处切开以暴露髌骨。从暴露的髌骨中去除约1mm厚的软骨,并用牙钻在股骨的凹槽中形成三个骨软骨缺损(直径2mm以及深度3mm)。将先前制备的各植入物植入所产生的缺损部位并结束手术,注射曲马多(5mg/kg)和土霉素(20mg/kg),然后在正常条件下饲养。
6周后,处死每只兔子,并从其膝盖区域取出股骨,然后使用10%中性缓冲福尔马林溶液(pH 7.4,BBC Biochemical,美国,华盛顿州弗农山)固定5天。用0.5%EDTA溶液处理固定的股骨以去除钙质,然后包埋在石蜡中。将包埋的股骨切碎以获得厚度为4μm的组织切片,并对获得的组织切片进行H&E染色和番红O染色(图7)。
另外,将组织切片浸入3%过氧化氢甲醇溶液中30分钟以抑制内源性过氧化物酶的活性,然后向其中添加蛋白酶K(Sigma-Aldrich,密苏里州,美国)并在37℃下使其反应10分钟。在反应结束后,用Ⅱ型胶原蛋白抗体(Calbiochem,加利福尼亚州,美国,稀释度1:100)对组织切片进行染色,然后用Vectastain Elite ABC-过氧化物酶试剂盒(VectorLaboratories Inc.,加利福尼亚州,美国)进行免疫染色。至于抗体反应,使用Vector SG(Vector Laboratories Inc.,加利福尼亚州,美国)来进行显色,并使用核快速溶液(Vector Laboratories Inc.,加利福尼亚州,美国)来进行复染(图7)。
图7是示出对将PLGA支架(PLGA scaffold)、在PLGA支架上培养synMSC而得的培养物(PLGA/SynMSC)以及在载有BMP-7的PLGA支架上培养synMSC而得的培养物(PLGA/SynMSC/BMP-7)移植的兔关节的股骨进行H&E染色、番红O染色和利用Ⅱ型胶原蛋白抗体的免疫染色的结果的显微照片。
如图7所示,在没有治疗受损部位的对照组的情况下,软骨层没有再生,其仅被纤维组织覆盖,并且未进行番红O染色。然而,在植入PLGA支架植入物的部位形成了软骨层,但是由于包含少量软骨细胞而形成了薄的软骨层,并且还形成了少量的蛋白聚糖。另外,确认到在PLGA支架上培养synMSC而得的培养植入物所植入的部位处,细胞水平有所提高并且在表面上再生了软骨。最后,证实了在载有BMP-7的PLGA支架上培养synMSC而得的培养植入物的所植入的部位处,再生了厚的软骨层并且还存在大量的Ⅱ型胶原蛋白。
因此,可知synMSC表现出使受损的骨和软骨再生的治疗效果并且该治疗效果由BMP-7提升。
根据以上描述,本发明所属领域的普通技术人员将理解,可以在不改变其技术构思或必要特征的情况下通过其他具体形态来实现本发明。鉴于此,上述实施例应当理解为在所有方面都是示意性的而不是限制性的。本发明的范围应被解释为包括从所附的权利要求的含义和范围及其等同概念(而不是上面的具体实施方式)得出的所有改变或修改。
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<120> 滑膜来源间充质干细胞及其用途
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Claims (11)
1.一种制备滑膜来源间充质干细胞的方法,包括:
(a)使滑膜组织包埋在水凝胶中并进行培养以获得培养物;以及
(b)通过在所获得的培养物中分解水凝胶来回收从所述滑膜组织迁移至水凝胶中并增殖的间充质干细胞。
2.如权利要求1所述的制备滑膜来源间充质干细胞的方法,其中所述水凝胶由选自由胶原蛋白(collagen)、明胶(gelatin)、软骨素(chondroitin)、透明质酸(hyaluronicacid)、藻酸(alginic acid)、基质胶(MatrigelTM)、壳聚糖(chitosan)、肽(peptide)、纤维蛋白(fibrin)、聚乙醇酸(PGA)、聚乳酸(PLA)、聚乙二醇(PEG)、聚丙烯酰胺(polyacrylamide)以及它们的组合组成的组中的物质构成。
3.如权利要求1所述的制备滑膜来源间充质干细胞的方法,其中通过使用选自由胶原酶、明胶酶、尿激酶、链激酶、组织纤溶酶原激活剂(TPA)、纤溶酶(plasmin)、透明质酸酶以及它们的组合组成的组中的酶进行处理来执行所述水凝胶的分解。
4.一种滑膜来源间充质干细胞,其通过如权利要求1至权利要求3中任一项所述的制备滑膜来源间充质干细胞的方法来制备且具有在细胞表面上表达CD29、CD44、CD73和CD90的免疫学特性。
5.如权利要求4所述的滑膜来源间充质干细胞,其能够分化为选自由脂肪细胞、骨细胞、软骨细胞及它们的组合组成的组中的细胞。
6.一种用于治疗骨损伤或软骨损伤的药物组合物,包含如权利要求4所述的滑膜来源间充质干细胞、其培养物或从所述间充质干细胞分化的细胞作为活性成分。
7.如权利要求6所述的用于治疗骨损伤或软骨损伤的药物组合物,其中所述滑膜来源间充质干细胞具有与支架混合的形态。
8.如权利要求7所述的用于治疗骨损伤或软骨损伤的药物组合物,其中所述支架是PLGA支架。
9.如权利要求6所述的用于治疗骨损伤或软骨损伤的药物组合物,还包含BMP-7。
10.一种用于培养间充质干细胞的组合物,包含滑膜组织和水凝胶。
11.一种用于培养间充质干细胞的试剂盒,包含如权利要求10所述的用于培养间充质干细胞的组合物。
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Publication number | Priority date | Publication date | Assignee | Title |
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CN112595698A (zh) * | 2020-11-18 | 2021-04-02 | 上海中医药大学 | 油红o的应用及定量检测组织或细胞内脂质的方法 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060160734A1 (en) * | 2003-11-26 | 2006-07-20 | Akihiko Kusanagi | In situ method for treatment and repair of meniscal injuries |
KR20090085806A (ko) * | 2008-02-05 | 2009-08-10 | 동국대학교 산학협력단 | 지방기원 중간엽 줄기세포를 연골세포로 분화시키는 방법 |
US20100136114A1 (en) * | 2006-07-10 | 2010-06-03 | The Trustees Of Columbia University In The City Of New York | De novo formation and regeneration of vascularized tissue from tissue progenitor cells and vascular progentitor cells |
CN103131675A (zh) * | 2013-02-07 | 2013-06-05 | 中国人民解放军第二军医大学 | 体外腺病毒介导bmp-2/7基因共表达诱导滑膜间充质干细胞向软骨细胞分化的方法 |
KR20130124074A (ko) * | 2012-05-04 | 2013-11-13 | 인제대학교 산학협력단 | 위장관, 말초신경, 또는 혈관 기원의 중간엽줄기세포의 배양방법 및 그 용도 |
WO2014088205A1 (ko) * | 2012-12-03 | 2014-06-12 | 사회복지법인 삼성생명공익재단 | 양성 연골종양세포를 이용하여 제조한 연골기질 및 이의 제조방법 |
US20160166734A1 (en) * | 2013-08-01 | 2016-06-16 | Two Cells Co., Ltd. | Cartilage-damage treatment agent and method for producing same |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6835377B2 (en) | 1998-05-13 | 2004-12-28 | Osiris Therapeutics, Inc. | Osteoarthritis cartilage regeneration |
CN1498266A (zh) | 2001-01-30 | 2004-05-19 | 使用滑膜衍生的组织或细胞治疗及修复关节软骨缺损或损伤的组合物及方法 | |
WO2008023829A1 (en) * | 2006-08-22 | 2008-02-28 | National University Corporation Tokyo Medical And Dental University | Application of synovium-derived mesenchymal stem cells (mscs) for cartilage or meniscus regeneration |
KR100902569B1 (ko) * | 2007-01-19 | 2009-06-11 | 재단법인서울대학교산학협력재단 | 인간 탯줄 유래 중간엽 줄기세포 및 이의 확립방법 |
JP5373427B2 (ja) | 2009-02-24 | 2013-12-18 | 国立大学法人 千葉大学 | 滑膜細胞および細切軟骨片の軟骨修復における使用 |
US9155607B2 (en) * | 2011-11-16 | 2015-10-13 | Purdue Research Foundation | Compositions and methods for repair or regeneration of soft tissue |
KR101613478B1 (ko) * | 2014-09-22 | 2016-04-19 | (주)안트로젠 | 중간엽줄기세포-하이드로겔을 함유하는 조성물 및 이의 제조방법 |
-
2019
- 2019-09-27 JP JP2021543107A patent/JP7230219B2/ja active Active
- 2019-09-27 EP EP19867525.8A patent/EP3865571A4/en active Pending
- 2019-09-27 CN CN201980078739.5A patent/CN113454206A/zh active Pending
- 2019-09-27 US US17/280,003 patent/US20220112465A1/en active Pending
- 2019-09-27 KR KR1020190119759A patent/KR102223043B1/ko active IP Right Grant
- 2019-09-27 WO PCT/KR2019/012602 patent/WO2020067774A1/ko unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060160734A1 (en) * | 2003-11-26 | 2006-07-20 | Akihiko Kusanagi | In situ method for treatment and repair of meniscal injuries |
US20100136114A1 (en) * | 2006-07-10 | 2010-06-03 | The Trustees Of Columbia University In The City Of New York | De novo formation and regeneration of vascularized tissue from tissue progenitor cells and vascular progentitor cells |
KR20090085806A (ko) * | 2008-02-05 | 2009-08-10 | 동국대학교 산학협력단 | 지방기원 중간엽 줄기세포를 연골세포로 분화시키는 방법 |
KR20130124074A (ko) * | 2012-05-04 | 2013-11-13 | 인제대학교 산학협력단 | 위장관, 말초신경, 또는 혈관 기원의 중간엽줄기세포의 배양방법 및 그 용도 |
WO2014088205A1 (ko) * | 2012-12-03 | 2014-06-12 | 사회복지법인 삼성생명공익재단 | 양성 연골종양세포를 이용하여 제조한 연골기질 및 이의 제조방법 |
CN103131675A (zh) * | 2013-02-07 | 2013-06-05 | 中国人民解放军第二军医大学 | 体外腺病毒介导bmp-2/7基因共表达诱导滑膜间充质干细胞向软骨细胞分化的方法 |
US20160166734A1 (en) * | 2013-08-01 | 2016-06-16 | Two Cells Co., Ltd. | Cartilage-damage treatment agent and method for producing same |
Non-Patent Citations (3)
Title |
---|
JACLYN LOCK等: "Nanophase hydroxyapatite and poly(lactide-co-glycolide) composites promote human mesenchymal stem cell adhesion and osteogenic differentiation in vitro", J MATER SCI: MATER MED, 7 July 2012 (2012-07-07) * |
JIABING FAN等: "In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels", ACTA BIOMATERIALIA, 19 July 2009 (2009-07-19) * |
TIAGO FERRO等: "Successful isolation and ex vivo expansion of human mesenchymal stem/stromal cells obtained from different synovial tissue‐derived (biopsy) samples", J CELLULAR PHYSIOLOGY, vol. 234, no. 4, 26 August 2018 (2018-08-26) * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112595698A (zh) * | 2020-11-18 | 2021-04-02 | 上海中医药大学 | 油红o的应用及定量检测组织或细胞内脂质的方法 |
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