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目的:探讨体外诱导人骨髓间充质干细胞(hMSCs)分化过程中心肌细胞特征表达及膜电位变化的情况,为hMSCs临床移植治疗提供实验参考数据。方法:采用体外纯化、扩增后的第4代hMSCs在体外经5μmol/L5-杂氮胞苷诱导24h后继续培养8周,相差显微镜下观察细胞形态变化,免疫组化方法鉴定心肌特异性蛋白心房钠尿肽(ANP)和连接蛋白(CX43)的表达,应用RT-PCR技术分析肌球蛋白重链(β-MHC)和肌钙蛋白T(cTnT)等相关基因在分化过程中的动态表达,全细胞膜片钳技术检测不同时期细胞膜电位的变化。结果:hM-SCs诱导前为纺锤形,诱导后第2天部分细胞即开始发生形变,呈球形或短棒状,1周后细胞质中颗粒增多,20%~30%细胞呈毛刷样变化;ANP和CX43在诱导前无表达,诱导后第2周开始表达,且表达随时间逐渐增强,β-MHC和cTnT mRNA分别在诱导后第1和4周时表达开始增强,在第6周时均达到高峰,第8周时表达开始衰减,hMSCs经诱导后随心肌样细胞特征的表达膜静息电位、去极化幅值和去极化速率逐渐增高。结论:hNSCs在体外经纯化、扩增和诱导后可表现心肌样细胞的生物学特性,可为hMSCs临床移植治疗提供可靠细胞来源,但移植后有形成心律失常的潜在危险。
OBJECTIVE: To investigate the characteristics of cardiomyocytes and the change of membrane potential during the differentiation of human mesenchymal stem cells (hMSCs) induced in vitro and to provide experimental reference data for the clinical transplantation of hMSCs. Methods: The hMSCs of passage 4 were purified and expanded in vitro. The hMSCs were further cultured for 8 weeks in vitro induced by 5μmol / L 5-azacytidine. The morphological changes of cells were observed under phase contrast microscope. The expression of myocardial specific protein was identified by immunohistochemistry Atrial natriuretic peptide (ANP) and connexin (CX43) were detected by RT-PCR. The dynamic expression of myosin heavy chain (β-MHC) and troponin T (cTnT) Whole cell patch clamp technique was used to detect the changes of cell membrane potential in different periods. RESULTS: The hM-SCs were spindle-shaped before induction. Some of the cells began to deform on the 2nd day after induction, and were spherical or short rod-shaped. After 1 week, the number of cytoplasm increased, and 20% -30% And CX43 did not express before induction, and began to express at the second week after induction, and the expression gradually increased with time. The expression of β-MHC and cTnT mRNA began to increase at 1 and 4 weeks after induction, respectively, reaching the peak at 6 weeks, At the 8th week, the expression began to decline. After hMSCs were induced, the resting membrane potential, the amplitude of depolarization and the rate of depolarization increased gradually with the cardiomyocyte-like characteristics. CONCLUSION: The hNSCs can express the biological characteristics of cardiomyocyte-like cells after purified, expanded and induced in vitro and provide a reliable source of cells for clinical transplantation of hMSCs. However, there is a potential risk of arrhythmia after transplantation.