论文部分内容阅读
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 The iPSCs derived from mice were resuscitated and cultured in hanging drop culture to form embryoid bodies. Vitamin C was used to induce the differentiation of iPSCs. The induction process was divided into electrical stimulation group and non-electrical stimulation group according to whether electrical stimulation was given or not. The growth of cells in each group was observed. The time of beating and the number of beating embryoid bodies were recorded and the rate of cardiomyocyte differentiation was calculated. Confocal microscopy and immunofluorescence cytochemistry were used to detect the expression of cardiac troponin T (c-TnT). RT-PCR was used to detect the gene expression of pluripotency markers Oct-4, GATA-4 and α-MHC. Results Compared with the non-electro-stimulated group, the electrical stimulation induced embryoid body differentiation faster and earlier pulsation. The rate of cardiomyocyte differentiation was higher (68.89 ± 5.09% vs 52.22 ± 3.85%, P <0.05). The c-TnT was expressed in both pulsatile cells in both groups, and the expression of c-TnT in electrical stimulation group was more obvious. During induction, the expression of Oct-4 gradually decreased with prolongation of induction time, and decreased more rapidly in electro-stimulation group than in non-electro-stimulation group (P <0.05). The expression of cardiac-specific marker GATA-4 and α-MHC were detected in both groups. At the same time point, the expression of GATA-4 and α-MHC in electrical stimulation group was higher than that in non-electrical stimulation group (P <0.05). Conclusion Electrical stimulation simulating the electrical cardiac microenvironment contributes to the differentiation of vitamin C-induced iPSCs into cells with phenotypic characteristics of cardiomyocytes in vitro.