仿生电刺激体外对ips细胞向心肌细胞诱导分化的 - 第三军医大学学报.doc

仿生电刺激对诱导多能干细胞体外向心肌细胞分化的作用 代艳梅，秦俭*，张晓刚，张学辉，陈川 (重庆医科大学附属第一医院心内科，重庆 400016) 第一作者简介：代艳梅，女，硕士研究生 医师 主要从事诱导多能干细胞向心肌细胞分化方面的研究 电话Email：dymme2006@126.com [摘要] 目的 探讨仿生电刺激在体外对诱导多能干细胞（iPSCs）向心肌细胞诱导分化的作用。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，诱导过程中按是否给予仿生电刺激分为两组。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。免疫荧光共聚焦成像检测心肌特异性肌钙蛋白c-TnT的表达。PR-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重链α-MHC的基因表达。结果 仿生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%）,P0.05]。两组搏动区域细胞均表达心肌特异性肌钙蛋白c-TnT。仿生电刺激组细胞骨架更清楚，胞浆内肌丝成束整齐排列；非电刺激组肌丝结构不清楚，体现为较幼稚的肌细胞形态。在诱导过程中，Oct-4表达水平随诱导时间延长逐渐减少，仿生电刺激组较非电刺激组递减更快（P0.05）。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，仿生电刺激组GATA-4和α-MHC表达水平高于非电刺激组（P0.05）。结论 模拟心脏电学微环境的仿生电刺激有助于维生素C诱导的iPSc在体外向功能完备的心肌细胞分化。 [关键词] 仿生电刺激；诱导多能干细胞；心肌细胞；细胞分化 [中图法分类号］ R-331 ［文献标识码］ A Effects of bionic electrical stimulation on the differentiation of induced pluripotent stem cells into cardiomyocytes induced by vitamin C in vitro ________________________ [通信作者] *通讯作者：秦俭 Tel：023 E-mail：hjqj2001@ Dai Yanmei, Qin Jian*, Zhang Xiaogang, Zhang Xuehui, Chen Chuan (Department of Cardiology，First Affiliated Hospital，Chongqing Medical University，Chongqing，400016，China)[Abstract] Objective To investigate the effects of bionic electrical stimulation on the differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocyte in vitro. Methods Thaw the vial of iPSCs derived from mouse. Use the classical hanging-drop method to make iPSCs form embryoid bodies（EBs）. Vitamin C was added during the entire differentiation period. The induced iPSCs were divided to stimulated group and non-stimulated group. Morphological changes of the EBs were observed dynamically.Record the time of contracting EBs appeared and numbers of contracting EBs in each group. The percentage of contracting EBs was calculated as the differentiation rate of cardiomyocyte. Expression of cardiac troponin I(cTnI) was observed by con