成年鼠骨髓间充质干细胞体外诱导分化为心肌样细胞

Induced differentiation of bone marrow mesenchymal stem cells into cardiomyocytes-like of adult mice in vitro

目的探讨成年大鼠骨髓间充质干细胞(MSC)体外培养条件及定向分化为心肌样细胞的可行性。方法通过取成年SD大鼠股骨干骨髓,进行体外培养和传代,用10μmol/L 5-氮杂胞苷(5-aza)分别对原代、一代和三代MSC进行体外定向诱导,用免疫组化方法分析检测分化后细胞的心肌特异性抗原(肌丝蛋白、肌钙蛋白)并通过RT-PCR鉴定心肌细胞特异因子基因(ANP、GATA-4)的表达。结果原代、一代MSC在体外经5-aza诱导4周后,细胞形态无明显变化,而经5-aza诱导4周后的三代MSC形态变大,呈杆形、球形,诱导细胞相互连接,连接细胞出现肌管样结构。免疫组化检测经10μmol/L 5-aza诱导4周的原代、一代MSC未见肌丝蛋白、肌钙蛋白T阳性细胞,而诱导三代MSC肌丝蛋白阳性比例近20%,肌钙蛋白阳性比例约8%。RT-PCR显示诱导三代MSC 4周后有ANP和GATA-4心肌细胞特异基因表达。结论MSC能够在体外经5-aza诱导分化为心肌样细胞,具有向心肌细胞转化的潜力,是自体心肌细胞移植的一种良好供体。

Objective To investigate the factors governing the growth and possibility that induced MSCs of adult mice to differentiate into cardiomyocytes-like in vitro. Methods Bone marrow mesenchymal stem cells were aspirated from adult SD mice,s femal bone and isolated by gradient centrifugation method. The MSCs （primary,first and third passage） were respectively treated with 5-aza for 24 h. The MSCs differentiation was observed by phase-contrast microscope, cardiac-specific antigen of the diffrentiated cells were detected by using immunohistochemistry, the expression of cardiomyocytes-specific gene was evaluated by reverse transcriptase-polymerase chain reaction （RT-PCR）. Results The MSCs of primary and first passages induced by 5-aza for 4 weeks showed no positive staining for Desmin and Troponin T, but the MSCs of the third passages induced by 5-aza for 4 weeks showed postive staining for Desmin and Troponin T. Desmin positive cells were 20% and Troponin T positive cells were nearly 8%. The MSCs of the third passage induced by 5-aza for 4 weeks expressed cardiac-special genes, including ANP, GATA-4. Conclusions The MSCs cultured with 5-aza could differentiate into cardiomyocytes-like in vitro, possessing as ideal dornor cells for implantation to myocardial infarction area.