体外培养鼠骨髓间充质干细胞及其向心肌样细胞诱导分化的实验研究

Proliferation of MSCs of mice in vitro and 5-azacytidine induces the differentiation of MSCs into cardiomyogenic cells

目的通过体外培养骨髓间充质干细胞(MSCs),并用5-氮胞苷诱导其向心肌样细胞分化,研究MSCs生物学特性和5-氮胞苷效能。方法将SD大鼠的骨髓采用贴壁法进行分离培养及传代增殖,相差显微镜下观察细胞形态变化,绘制生长曲线,并用流式细胞仪技术检测CD29、CD45的表达,进行细胞成分鉴定。用5-氮胞苷体外诱导MSCs,观察其形态结构。用免疫组化检测肌钙蛋白T(cTnT)、结蛋白(Desmin),用RT-PCR检测心肌特异因子基因β-肌球蛋白重链(β-MHC)的表达。结果贴壁法分离培养的MSCs生长稳定,增殖较快。经5-氮胞苷诱导后,MSCs形态发生变化,可见肌管样结构。免疫组化和RT-PCR证实MSCs经体外诱导发生了向心肌样细胞的分化。结论采用贴壁筛选法,简便易行,短期内可获得大量的纯度较高的骨髓间充质干细胞;在5-氮胞苷的诱导下,骨髓间充质干细胞呈现向心肌样细胞分化的趋势。

Objective To investigate the biological characteristics and cardiomyogenic potential of MSCs in vitro. Methods MSCs were isolated and parified by the attachment treatment,then cultured in IMDM medium with 10% fetal bovine serum. The biological characteristics and growth effects of the cells were analyzed. C1Y29 and CD45 were detected by immunofluorescence flow cytometry,which were used to identify the cell components. Mesenchynml stem cells （MSCs） which incubated with 5-azacytidine were induced into cardiomyogenlc cells. Results MSCs were the cells collection with several sorts of cells,most of which were MSCs. FACS could analyse for CD29 and CD45,which showed that the cells were MSCs. After incubated with 5-azacytidine, MSCs could be induced into cardiomyogenic cells with typical morphological and histological characteristic. Conclusion The preplating method can be used for MSCs isolation. After treated with 5-azacytidine, the MSCs may be differentiated into cardiomyogenic cells in vitro.