MSC体外诱导分化为心肌样细胞的鉴定及Nesprin蛋白表达研究

Identification of cardiomyocytes differentiated from mesenchymal stem cells and study on expression of Nesprin protein

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 and identify the differentiation of rat bone marrow mesenchymal stem cells （MSCs） into cardiomyocytes in vitro, and observe the expression of Nesprin protein during the differentiation. Methods Rat MSCs were isolated and purified by Ficoll density gradient centrifugation, and adhered for serial subcuhivation. Surface-associated antigens of MSCs of the second passage were dedected by flow cytometry. MSCs of the second passage were induced by 10 μmol/L 5-azacytidine （5-Aza） to differentiate into cardiomyocytes, and the morphological changes were observed. The expression of Desmin, α-sarcomeric actin and cardiac Troponin I （cTnI） mRNA and protein was detected by RT-PCR, immunocytochemistry and immunofluorescence staining, and the expression of Nesprin protein was detected by Western blotting. Results The morphology of MSCs induced by 5-Aza was bigger and longer, and the nuclei became bigger, exhibiting more consistent patterns. The expression of Desmin, α-sarcomeric actin and cTnI mRNA and protein of MSCs was positive. Immunofluorescence staining revealed that Nesprin protein positioned in the nuclear membrane, and Western blotting detection demonstrated that the expression of Nesprin protein significantly increased after differentiation （P 〈 0.05）. Conclusion MSCs may be successfully induced to differentiate into cardiomyocytes. The expression of Nesprin protein in the differentiated MSCs may significantly increase, indicating Nesprin may play a role in the differentiation from MSCs to cardiomyocytes.