人骨髓间充质干细胞体外定向分化过程中心肌细胞特征表达及膜电位变化

Analysis of characteristics and electrophysiological properties during directional differentiation of human mesenchymal stem cells to cardiomyocytes in vitro

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 differentiation and electrophysiological properties during the process of differentiating of hMSCs into cardiomyocytes in vitro directionally and provide experimental data for clinical transplant therapy of hMSCs. Method： hMSCs were isolated and expanded as undifferentiated cells in culture until 4th passages, then were treated with 5 μmol/L 5-azacytidine for 24 hours and cultured for 8 weeks,The differentiation of hMSCs was observed by Phase-Contrast microscope,Immunohistochemistry and RT-PCR, Membrane potential were observed in the whole cell patch-clamp mode. Result：hMSCs had a spindle morphology,but formed a ball-like or strick-like morphology at 2 days after induction, 1 week later, approximately 30% of the cells gradually increased in size and granules were seen concentrating in cells. Atrial natriuretic peptide（ANP） and Connexin43 （CX43） began to express at 2nd week after induction and increased gradually. About 60% of the resulting myogenic cells were positive at 4th week after induction. It＇s negative for uninduced cells. The expresstion level of β-MHC and cTnT mRNA were undetectable before induction, and began to increase at 1、4 weeks after induction, then reached the peak at 6th week and decreased after that. hMSCs＇resting membrane potential（RMP）, range of depolarization and rate of depolarization were heightened increasely after be induced. Conclusion： hMSCs can express biological characteristics of cardiomyocytes pbenotype cells after be purified, expanded, induced in vitro, and provide reliable cells for clinical transplant therapy. But they may be the sources of unanticipated arrythmias after cell transplantation.