体外模拟心肌微环境中人骨髓间充质干细胞向心肌细胞的分化

Effect of simulated cardiac microenviroment in vitro on differentiation of human marrow mesenchymal stem cells to cardiomyocytes

目的:研究在体外模拟心肌微环境中人骨髓间充质干细胞(hMSCs)向心肌细胞(CM)的分化。方法:体外分离培养扩增hMSCs,进行流式细胞仪分析鉴定其免疫学表型CD34、CD44和CD45,以未加一抗只加二抗的hMSCs作为平行对照组;同时原代培养SD乳鼠的CM以模拟体外心肌微环境。选用生长良好、纯度达到95%的第5代hMSCs,与乳鼠原代培养的CM共培养,并进行心肌特异性标志cTnI的免疫组化鉴定。结果:体外分离纯化培养扩增出的hMSCs,不表达CD34及CD45,高表达CD44,其CD44阳性率平均为(93.26±2.48)%,与平行对照组[(3.42±1.09)%]比较差异有显著性(P<0.01)。hMSCs与CM共培养后,其形态逐渐向CM形态过渡,并表达心肌特异性抗体cTnI。结论:hMSCs在体外模拟的心肌微环境中可分化为CM,干细胞的分化具有环境依赖性。

Objective To study the effect of simulated cardiac microenvironment in vitro on the differentiation of human marrow mesenchymal stem cells （hMSCs） to cardiomyocytes （CM）. Methods hMSCs were isolated and expanded in culture and the phenotypes （CD34, CD44 or CD45） of hMSCs were identified by fluorescent - activated cell sorting （ FACS ）. The parallel control groups were without mono-antibody. Fifth passage hMSCs, having good growth characteristics and the purity〉 95%, were co-cultivated with CM, which derived from 2-day neonatal SD rats as simulating of the cardiac microenvironment. Expression of cardiac-specific marker cTnI, identified by immunohistochemistry, was used as a marker of cardiac muscle differentiation. Results hMSCs did not express CD34 and CD45; but it expressed a high level of CD44, the positive rate of CD44 in hMSCs group （93.26%± 2.48%） was higher than that in parallel control group （3.42%±1.09%） （P〈0.01）. After co-culture of hMSCs and CM, the morphology of hMSCs changed closely into that of CM, and the cells demonstrated positive staining for cTnI. Conclusion hMSCs have the potential to differentiate into cardiomyocytes under the simulated cardiac microenvironment in vitro.