鼠胚胎干细胞早期分化来源的心肌细胞同种异体移植后的电生理特性

目的 探讨小鼠胚胎干细胞早期分化来源的心肌细胞进行同种异体移植后的电生理特性.方法 首先通过电转染构建a-MHC-EGFP-ESc系[将心肌细胞特异的OL-MHC启动子与表达基因--绿色荧光蛋白(EGFP)基因融合,构建成真核表达载体],悬滴法诱导胚胎干细胞分化,在分化早期(7+4)d利用流式细胞仪筛选带绿色荧光的心肌细胞,将纯化的心肌细胞(5×106个/ml)移植到小鼠心室壁,对照组注入等体积培养基,移植前3 d开始应用环孢素A(静脉注射,5 mg·kg-1·d-1)和泼尼松龙(静脉注射,2.5 mg·kg-1·d-1)抑制免疫排斥反应.移植后2周分离两侧颈迷走神经行电刺激抑制窦房结与房室结,记录刺激前后的体表心电图,然后分别行免疫荧光显像和膜片钳研究.结果 刺激迷走神经前,移植组和对照组均呈正常的窦性心律,两组无室性心律失常的发生;刺激迷走神经后,两组动物均出现异位的心室起搏心律,细胞移植组与对照组心室频率无差异.移植区冰冻切片免疫荧光分析可见EGFP标记的移植细胞具有肌钙蛋白I(cTnI)表达,说明分化细胞移植后仍具有心肌特性,且移植细胞与宿主心肌细胞间有连接蛋白43的表达,表明移植细胞与宿主心肌细胞间形成电偶联通道.膜片钳分析,绿色荧光细胞移植前后其具有起搏细胞动作电位的比例分别为(85.1%vs 1 1.4%,P＜0.05),该类细胞在移植前后的起搏电流强度有所增强((11.2±2.4)pA/pF vs(15.5±1.9)pA/pF,P＜0.05].移植区分离的绿色荧光细胞中48%具有心室肌动作电位.结论 胚胎干细胞早期分化来源的心肌细胞在体移植后能进一步分化为成熟的心室肌细胞及起搏细胞.

The propriety of early differentiated cardiomyocytes from embryonic stem cells after transplantation

Objective Cell transplantation has been proposed as a potential approach for repairing damaged myocardium.The present study investigated the propriety of early differentiated cardiomyocytes from embryonic stem cell(ESc)after transplantation.Methods At early stage of differentiation,cardiomyocytes from ESc were selected by fluorescence activated cell sorting,and then were transplanted into the ventricular of mice.The experimental mice were separated into two groups,the control group and the cell-transplanted group.Two weeks after cells injection,mice were subjected to vagal stimulation to permit emergence of escape pacemakers.Electrocardiography was used.Then the injected cardiac tissue was seized,single myocyte was obtained by digestion,patch clamp technique was used to test It and action potential.Results Sustained electrocardiography recording revealed a stable idioventricular rhythm.The transplanted cells exhibited significantly increased If current than before transplantation.The most transplanted cells which present pacemaker-like action potential had the ventricular-like action potential after transplantation.Conclusion Early differentiated cardiomyocyte derived from mESc can continue to development in electrophysiological characteristic after transplantation.