骨髓间充质干细胞体外诱导分化为心肌样细胞的实验研究

Experimental Study on Differentiation of Bone Marrow Mesenchymal Stem Cells into Cardiomyocyte-like Cells in Vitro

目的探讨骨髓间充质干细胞(mesenchymal stem cells,MSCs)是否能体外诱导分化为心肌样细胞。方法用浓度梯度法分离和纯化Wistar大鼠MSCs,贴壁法培养,培养的第2代细胞分别用终浓度为0、0.1、1.0、10.0、50.0、100.0μmol/L的5-氮杂胞苷(5-azacytidine,5-aza)诱导分化,用光学显微镜观察细胞的形态并用免疫细胞化学方法鉴定细胞内的心肌肌钙蛋白I(cardiac troponinI,cTnI)。结果以终浓度10.0μmol/L5-aza为诱导组,细胞从原来的梭形变为排列趋向较为一致的长梭形,且体积增大,诱导10d后,细胞内cTnI蛋白染色阳性。终浓度为0、0.1、1.0μmol/L组细胞形态变化不明显,细胞内cTnI染色阴性。终浓度为50.0、100.0μmol/L组细胞死亡。结论大鼠MSCs细胞可经5-aza诱导分化为心肌样细胞,且5-aza的最佳诱导浓度为10.0μmol/L。

Objective To observe the differentiation of bone marrow MSCs into cardiomyocyte-like in vitro. Methods MSCs were isolated from bone marrow of rat by density gradient centrifugation and anchoring culture. MSCs of passage 4 were induced to differentiate by different concentration, including 0,0. 1,1.0,10.0,50.0 and 100.0μmol/L, of 5-azacytidine（5-aza） in culture medium. The morphology of the cells was observed by optical microscope, and, the differentiation was identified by immunochemistry technique. Results In group of 10μmol/L 5-aza,the MSCs were found to change their round shape into spindle shape, arraying in similar direction gradually. cTnI was detected at 10 days after inducted by 5-aza. But in groups of 0, 0.1 or 1.0μmol/L 5- aza, the MSCs had no changes in morphology,and cTnI cannot be detected. In groups of 50.0 and 100.0μmol/L, the cell died. Conclusion MSCs can be induced to cardiomyocyte-like cells by 5-aza, and the optimal inducing concentration of 5-aza is 10.0μmol/L.