心肌细胞共培养诱导骨髓间充质干细胞向心肌样细胞分化

Co-culture of cardio myocytes induces mesenchymal stem cell to diffenertiate into cardiomyocyte-like cells

目的在体外以新生大鼠心肌细胞(CM)与骨髓间充质干细胞(MSCs)共同培养的方式模拟心肌微环境,研究MSCs分化为心肌细胞的机制。方法分离大鼠MSCs在体外培养纯化后进行细胞标记,将已标记的MSCs分别与搏动的CM、停止搏动的CM以及心肌细胞条件培养液混合培养。分别在共培养后第4、5天用免疫荧光染色检测MSCs细胞中的心肌特异性肌钙蛋白T(Troponin T)。结果与搏动的CM共培养后第4天MSCs出现自发收缩,与CM同步搏动并表达Troponin T,而在抑制心肌细胞搏动或缺乏与心肌直接接触的情况下MSCs未出现上述变化。结论说明在与CM直接接触的前提下,CM对MSCs的机械牵拉刺激为诱导MSCs分化为心肌细胞的必须条件。单纯的心肌细胞条件培养液则非关键因素。了解MSCs分化为心肌细胞的机制对于寻找适宜的细胞移植条件有重要指导意义。

Objective To explore the related mechanisms of mesenchymal stem cells （MSCs） differentiating into cardiomyocytes（CM） in a simulated cardiac micro-environment. Methods After labelled with 4,6-dimidino-2-phenylindone （DAPI）, MSCs were seperately cocuhured in direct cell-cell contaction with beating CM and non-beating CM whose spontaneous beatings were inhibited. In addition, MSCs were also cultured in the presense of media conditioned by separate cultures of CM. Then MSCs were stained for the expression of troponin T on the fourth, fifth day respectively. Results When cocuhured with beating CM, MSCs started to contract synchronously with CM on fourth day and were identified by the positive staining for Troponin T. However both physical contract and the expression of Troponin T protein in MSCs were not found in other groups. Conclusion Direct cell-cell contact and mechenical force of beating CM were necessary for transdifferentiation of MSCs into cardiomyocytes in vitro, but conditioned media is not sufficient for the differentiation.