体外电刺激对维生素C诱导的多能干细胞向心肌细胞分化的影响  

Effect of electrical stimulation on the differentiation of induced pluripotent stem cells into cardiomyocytes induced by vitamin C in vitro

作　　者：代艳梅[1] 秦俭[1] 张晓刚[1] 张学辉[1] 陈川[1] 廖康腊[1] 

机构地区：[1]重庆医科大学附属第一医院心内科,重庆400016

出　　处：《细胞与分子免疫学杂志》2013年第4期364-367,371,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：重庆市卫生局医学科学技术研究重点项目(2009-1-51)

摘　　要：目的探讨电刺激在体外对诱导性多能干细胞(iPSCs)向心肌细胞分化的作用。方法复苏小鼠来源的iPSCs,悬滴培养法形成拟胚体,维生素C诱导其分化,诱导过程按是否给予电刺激分为电刺激组与非电刺激组。观察各组细胞生长情况,记录各组拟胚体出现跳动的时间和跳动拟胚体的数量,计算心肌细胞分化率。共聚焦显微镜成像结合免疫荧光细胞化学检测心肌特异性肌钙蛋白T(c-TnT)的表达。RT-PCR检测干细胞多能性标志因子Oct-4、心肌早期特异性转录因子GATA-4和心肌特异性肌球蛋白α重链(α-MHC)的基因表达。结果电刺激组拟胚体较非电刺激组分化更快,更早出现搏动,心肌细胞分化率更高[(68.89±5.09)%vs(52.22±3.85)%,P<0.05]。两组搏动区域细胞均表达c-TnT,电刺激组细胞c-TnT表达更明显。在诱导过程中,Oct-4表达水平随诱导时间延长逐渐减少,电刺激组较非电刺激组递减更快(P<0.05)。两组均检测到心肌特异性标记分子GATA-4和α-MHC的表达。在诱导分化相同时间点,电刺激组GATA-4和α-MHC表达水平高于非电刺激组(P<0.05)。结论模拟心脏电学微环境的电刺激有助于维生素C诱导的iPSCs在体外向具有心肌细胞表型特征的细胞分化。Objective To investigate the effect of electrical stimulation on the differentiation of induced pluripotent stem cells （iPSCs） into cardiomyocytes in vitro. Methods Classical hanging-drop method was used to induce iPSCs from mice to form embryoid bodies （EBs） and vitamin C was contained in the medium through the induction period. According to whether or not electrical stimulation was used in the whole induction period, iPSCs were divided to electrical stimulation group and non-stimulation group. During the induction, dynamic morphological changes of the EBs were observed and photographed, the time point when beating EBs in each group appeared was recorded and the number of them was counted. The percent- age of beating ones in all EBs was calculated as the differentiation rate of cardiomyocytes induced from iPSCs. Furthermore, expression of cardiac troponin T （cTnT） was observed by immunofluorescent staining under a confocal laser scanning micro-scope （CLSM）, and mRNA expression levels of the related genes Oct-4, GATA-4 and α-MHC were analyzed by RT-PCR. Results Compared with the non-stimulation group, beating cells in electrical stimulation group appeared in a shorter time, and the differentiation rate of cardiomyocytes was higher [ （68.89 ± 5.09 ） % vs （.52.22 ± 3.85 ） %, P 〈 0.05 ]. c-TnT was expressed in the beating area of both groups, but the cells in the electrical stimulation group showed a more clear cytoskeleton. The mRNA level of Oct-4 decreased in a time-dependent manner in the whole period of induction and in the electrical stimulated group it decreased faster than the non-stimulation group （P 〈0.05）. In addition, more GATA4 and α-MHC mRNA in electrical stimulation group were expressed than the non-stimulated group at the same point-in-time （ P 〈 0.05）. Conclusion The electrical stimulation which simulates cardiac electrical microenvironment to some extent improved the differentiation of iPSCs into functional cardiomyocytes induced by vitamin C in vitro.

关 键 词：电刺激 诱导多能干细胞 心肌细胞 分化 

分 类 号：R-331[医药卫生] R392-33