猪骨髓间充质干细胞体外向心肌细胞分化的实验研究

【作者】 闻松男；

【导师】 杨跃进；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 目的:探讨猪骨髓间充质干细胞(mesenchymal stem cells,MSCs)自发分化和5-氮胞苷诱导分化为心肌细胞的潜能,以及体外向心肌细胞分化的规律性。方法:抽取猪髂骨骨髓,密度梯度离心法分离单个核细胞,贴壁培养法筛选和培养MSCs,选取第9,10,11,12,15和20代MSCs,研究其向心肌样细胞自发分化潜能和5-氮胞苷诱导后4周能否分化为心肌细胞,并探索猪MSCs体外向心肌细胞分化的规律性。各代MSCs采用连续显微镜下观察、流式细胞仪、免疫细胞化学染色法、Real Time-PCR、透射电镜检测相关指标。结果:体外自然培养条件下,P9,P11,P12,P15,P20和大部分P10 MSCs形态一致,呈梭形成纤维细胞样形态,但部分P10细胞多核、胞浆延长,转变为肌管样形态。5-氮胞苷诱导1周后,部分P9,P11,P12,P15和P20MSCs表现为较长的胞浆形态和多核化,4周后更多细胞相互连接形成不规则的管样结构,而P10MSCs则形成相对规则的肌管样结构。流式法显示,P10MSCs的G0/G1期比例显著增多(88.00±3.90)%,P10与P9相比有显著性差异(P＜0.0001),出现细胞生长停滞。贴壁细胞CD90、CD29阳性,而CD34、CD45阴性,说明贴壁细胞为MSCs。免疫细胞化学染色显示,自然培养和诱导4周后各代MSCs均表达心肌特异性结构蛋白Cx43和α-sarcomeric actin,而诱导后P10MSCs分化率与其他各代相比有显著性差异(P=0.011)。Real time-PCR结果表明,自然培养和诱导4周后的各代MSCs均有心肌特异性转录因子GATA4和结构基因MLC、α-SKA、Cx43、cTNI基因表达,而P10MSCs自然培养和诱导后4周,各基因的表达丰度均高于其他代在相同条件下的分化细胞,而α-SKA诱导后增高显著(P＜0.0001),而P20MSCs表达的基因减少。透射电镜显示在自然培养状态下和诱导后4周,P9,P10,P11,P12,P15 MSCs均有较多不规则细肌丝,而P10MSCs经诱导后出现较多不规则排列粗肌丝,P20MSCs肌丝减少。结论:1.猪骨髓MSCs在体外自然培养条件下,各代形态、增殖能力和分化潜能是不均一的,传代次数的增加MSCs增殖逐渐变慢、部分细胞自发分化为心肌样细胞。2.MSCs于第10代出现细胞生长停滞,但并没有丧失多向分化潜能。3,在体外长期培养过程中,MSCs出现转化能力。4.5-氮胞苷可诱导MSCs分化为心肌样细胞,P10MSCs分化更具有成熟心肌细胞的特异性表型。4.结合本实验研究组前期的结果,推测P10可能是猪MSCs体外分化为心肌细胞过程中的拐点。更多还原

【Abstract】 Objectives:Mesenchymal stem cells(MSCs) are a group of heterogenous stem cells with multipotency in growth and differentiation.The aim of this study was to explore the proliferative potential and cardiomyogenic phenotype development of porcine bone marrow- derived MSCs before or after 5-azacytidine treatment,and find out the regularity of MSCs differentiation in vitro.Methods:Bone marrow samples were aspirated from Chinese mini-swine.The mononuclear cells were isolated by density gradient centrifugation through 1.077g/ml Percoll and then cultured in high-glucose DMEM containing 10%fetal bovine serum.After primary culture,relatively purified MSCs were obtained by using the method of adherence screening.MSCs at passage9,10,11,12,15 and 20 were examined for their proliferation and differentiation ability before or after 5-azacytidine(10μM) treatment in vitro by FACS,immunocytochemistry, transmission electrical microscopy and real time-PCR.Results:In normal growth medium,porcine bone marrow-derived MSCs were positive for CD90 and CD29,negative for CD34 and CD45.The growth property analysis demenstrated P10 pMSCs showed a growth-arrest appearance,while other passages displayed an exponential growth pattern.MSCs at every passage in vitro expressed cardimyocyte-specific genes and proteins.However,only passage 10 took on the formation of myotube structure.After 5- azacytidine treatment,pMSCs at every passage in vitro took on cardiomyocyte -like phenotype and formation of myotube structure,and expressed cardimyocyte-specific genes and proteins.However,MSCs at passage 10 expressed cardimyocyte-specific genes higher than any other passages,especiallyα-SKA,P10 vs P9,P10vs P11,P10vs P12,P10vsP15,P10vs P20(P＜0.0001).Before or after 5- azacytidine treatment,MSCs at passage 9,11,12,15 and 20 all had cardiomyocyte-like ultrastructure such as irregular myofilaments in vitro. However,only MSCs at passage 10 took on relatively mature cardiomyocyte ultrastructure,including myofilaments.MSCs had no observation of sarcomeres, transverse striations,intercalated discs in vitro.Conclusion:Cardiomyogenic potential of porcine bone Marrow-derived MSCs spontaneously or in response to stimulation of 5-azacytidine in vitro is dependent upon the endogenous gene expression and passage-restricted pattern.These findings reveal a novel regulation on the transdifferentiation of MSCs and provide useful information for exploiting the clinical therapeutic potential of MSCs.更多还原