H9C2细胞培养液诱导大鼠骨髓间充质干细胞向心肌样细胞分化

背景：文献报道间充质干细胞经体外化学药物诱导或自体移植体内诱导或模拟心肌样微环境体外诱导等方法可不同程度的诱导心肌细胞分化，但这些方法诱导率低、操作复杂、毒副作用大。 目的：验证心肌细胞株H9C2细胞培养液对骨髓间充质干细胞分化为心肌样细胞的诱导作用。 方法：运用全骨髓贴壁筛选法分离培养大鼠间充质干细胞，制备 H9C2细胞培养液作为诱导培养液，将间充质干细胞诱导培养1，3，5，7 d；以单独10%F12-DMEM培养液培养的H9C2细胞为阳性对照组；单独10%F12-DMEM培养液培养的间充质干细胞为阴性对照组。用免疫荧光法和western-blot检测其肌钙蛋白T、心肌细胞结蛋白的表达，用实时荧光定量检测其心肌细胞特征性基因α-肌球蛋白重链和β-肌球蛋白重链mRNA的表达。 结果与结论：H9C2细胞培养液诱导间充质干细胞培养7 d，间充质干细胞增殖分化细胞中肌钙蛋白T阳性细胞达（16±7）%，显著高于对照组（P 〈0.05）；与阴性对照组比较，western-blot检测诱导培养间充质干细胞后分化细胞肌钙蛋白T表达明显上调（P〈0.05），结蛋白表达明显上调（P 〈0.05）；RT-PCR检测分化细胞α-肌球蛋白重链与β-肌球蛋白重链mRNA表达均上调（P 〈0.05）。结果提示H9C2细胞培养液能诱导间充质干细胞向心肌样细胞分化。

Differentiation of mesenchymal stem cells into cardiomyocyte-like cells induced by H9C2 cell culture medium

BACKGROUND：Mesenchymal stem cels can differentiate into cardiomyocytesin vitro induced by different methods, such as chemical drug induction, autologous transplantationin vivo, andin vitro simulation of cardiac-like microenvironment, but these inducible methods show low induction rate, complex operation, and toxic side effects.
OBJECTIVE：To investigate the role of H9C2 cellculture medium in the differentiation of mesenchymal stem cels into cardiomyocyte-like cels.
METHODS： Mesenchymal stem cels were obtained by the whole bone marrow adherent culture and H9C2 cellculture medium was prepared as a culture medium. Then mesenchymal stem cels were co-cultured with H9C2 cellculture medium for 1, 3, 5, 7 days. H9C2 cels cultured in 10% Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 served as positive control groups, while mesenchymal stem cels cultured in 10% Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 as negative control group. Immunofluorescence and western blot assay were used to detect expression of myocardial celljunction protein （desmin） and troponin T. Real-time quantitative PCR was applied to detect mRNA expression of myocardial celltrait genes, α-cardiac myosin heavy chain and β-myosin heavy chain.
RESULTS AND CONCLUSION：After co-culture of H9C2 cellculture medium and mesenchymal stem cels for 7 days, the troponin T positive cels were up to （16±7）%, which was significantly higher than that of non-induced mesenchymal stem cels. Compared with the negative control group, the expression of troponin T protein and desmin after induction were significantly increased （P 〈 0.05） by western-blot detection; real-time PCR showed that the mRNA expression ofα-cardiac myosin heavy chain and β-myosin heavy chain in differentiated cels were both up-regulated （P 〈 0.05）. These findings suggest that H9C2 cellculture medium may induce the differentiation of mesenchymal stem cels into cardiomyocyte-like cels.