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目的体外诱导大鼠骨髓间充质干细胞(MSC)向心肌细胞分化并进行相关鉴定,观察分化过程中细胞Nesprin蛋白表达的变化。方法大鼠MSC经Ficoll密度梯度离心法分离获得后贴壁传代培养,流式细胞术鉴定第2代MSC表面抗原表达。以10μmol/L 5-氮杂胞苷(5-Aza)诱导第2代MSC向心肌细胞分化,观察细胞形态学改变;RT-PCR、免疫组织化学法和免疫荧光细胞化学染色鉴定心肌细胞相关蛋白Desmin、α-sarcomeric actin、cardiac TroponinⅠ(cTnⅠ)mRNA和蛋白表达;Westernblotting检测MSC分化前后Nesprin蛋白表达。结果经5-Aza诱导分化后的MSC细胞及细胞核增大,形态渐趋一致,大部分呈长梭形;细胞Desmin、α-sarcomeric actin、cTnⅠmRNA和蛋白呈阳性表达。免疫荧光细胞化学染色显示Nesprin蛋白定位于核膜,Western blotting分析发现诱导分化后细胞Nesprin蛋白表达明显增加(P<0.05)。结论成功诱导大鼠MSC向心肌细胞分化;Nesprin蛋白在分化后细胞中的表达明显增加,提示Nesprin可能参与MSC向心肌细胞样变化的过程。
OBJECTIVE: To induce the differentiation of rat bone marrow mesenchymal stem cells (MSC) into cardiomyocytes in vitro and to identify the changes of Nesprin protein expression during differentiation. Methods The rat MSC were isolated by Ficoll density gradient centrifugation and then subcultured. The surface antigens of the second generation MSCs were identified by flow cytometry. The second generation of MSCs was induced to differentiate into cardiomyocytes with 10μmol / L 5-azacytidine (5-Aza), and morphological changes of cells were observed. RT-PCR, immunohistochemistry and immunofluorescence staining were used to identify cardiomyocyte-associated protein Desmin, α-sarcomeric actin, cardiac troponinⅠ (cTnⅠ) mRNA and protein expression; Western blotting detected before and after NMSprin protein expression. Results After 5-Aza-induced differentiation, the number of MSC and nucleus increased, and the morphology became more consistent. Most of them showed long fusiform shape. The expression of Desmin, α-sarcomeric actin and cTnⅠ mRNA and protein were positive. Immunofluorescence staining showed that Nesprin protein was located on the nuclear membrane. Western blotting showed that Nesprin protein expression was significantly increased (P <0.05). Conclusion The differentiation of rat MSC into cardiomyocytes was successfully induced. The expression of Nesprin protein in the differentiated cells was significantly increased, suggesting that Nesprin may be involved in the process of cardiomyocyte-like changes.