CAJ | 학술논문

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.
목적 탐토소서배태간세포조기분화래원적심기세포진행동충이체이식후적전생리특성.방법 수선통과전전염구건a-MHC-EGFP-ESc계[장심기세포특이적OL-MHC계동자여표체기인--록색형광단백(EGFP)기인융합,구건성진핵표체재체],현적법유도배태간세포분화,재분화조기(7+4)d이용류식세포의사선대록색형광적심기세포,장순화적심기세포(5×106개/ml)이식도소서심실벽,대조조주입등체적배양기,이식전3 d개시응용배포소A(정맥주사,5 mg·kg-1·d-1)화발니송룡(정맥주사,2.5 mg·kg-1·d-1)억제면역배척반응.이식후2주분리량측경미주신경행전자격억제두방결여방실결,기록자격전후적체표심전도,연후분별행면역형광현상화막편겸연구.결과 자격미주신경전,이식조화대조조균정정상적두성심률,량조무실성심률실상적발생;자격미주신경후,량조동물균출현이위적심실기박심률,세포이식조여대조조심실빈솔무차이.이식구빙동절편면역형광분석가견EGFP표기적이식세포구유기개단백I(cTnI)표체,설명분화세포이식후잉구유심기특성,차이식세포여숙주심기세포간유련접단백43적표체,표명이식세포여숙주심기세포간형성전우련통도.막편겸분석,록색형광세포이식전후기구유기박세포동작전위적비례분별위(85.1%vs 1 1.4%,P＜0.05),해류세포재이식전후적기박전류강도유소증강((11.2±2.4)pA/pF vs(15.5±1.9)pA/pF,P＜0.05].이식구분리적록색형광세포중48%구유심실기동작전위.결론 배태간세포조기분화래원적심기세포재체이식후능진일보분화위성숙적심실기세포급기박세포.
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.