体外诱导心肌样细胞中HCN4过表达对起搏电流特性的影响  

Effect of HCN4 overexpression on the characteristics of pacemaker current in cardiomyocyte-like cells in vitro

作　　者：王红丽 李志强[2] 周贤惠[3] 李耀东[3] 汤宝鹏[3] WANG Hongli;LI Zhiqiang;ZHOU Xianhui;LI Yaodong;TANG Baopeng(Clinical Medical Research Institute,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China)

机构地区：[1]新疆医科大学第一附属医院临床医学研究院,乌鲁木齐830054 [2]新疆医科大学动物实验中心,乌鲁木齐830054 [3]新疆医科大学第一附属医院心脏起搏电生理科,乌鲁木齐830054

出　　处：《中华实用诊断与治疗杂志》2018年第10期945-948,共4页Journal of Chinese Practical Diagnosis and Therapy

基　　金：新疆维吾尔自治区自然科学基金(2014211C066)

摘　　要：目的体外构建具有表达功能性的起搏心肌样细胞,并检测起搏心肌样细胞中起搏电流的特性。方法分离培养大鼠骨髓间充质干细胞诱导分化为心肌样细胞。构建携带人HCN4基因的重组腺病毒载体(rAd/GFP/HCN4)和绿色荧光蛋白的腺病毒载体(rAd/GFP),转染至心肌样细胞分为rAd/GFP/HCN4组和rAd/GFP组,不做任何处理作为对照组。Western blot检测各组HCN4蛋白表达,全细胞膜片钳技术记录各组中起搏电流情况。结果荧光显微镜观察到长梭形心肌样细胞,PCR鉴定重组腺病毒载体rAd/GFP/HCN4和rAd/GFP构建成功;rAd/GFP/HCN4组HCN4蛋白相对表达量(1.31±0.02)高于rAd/GFP组(0.55±0.02)和对照组(0.52±0.03)(P<0.05),rAd/GFP组与对照组比较差异无统计学意义(P>0.05);膜片钳实验在转基因细胞中检测到起搏电流,而rAd/GFP组和对照组中未检测到起搏电流。结论 rAd/GFP/HCN4转染到心肌样细胞中,心肌样细胞中HCN4蛋白表达增加且可检测到起搏电流,从而使心肌样细胞具有自主搏动,为体外构建生物起搏器提供研究基础。Objective To construct pacing cardiomyocyte-like cells in vitro and to study the characteristics of pacemaker current in cardiomyocyte-like cells.Methods Bone marrow mesenchymal stem cells（BMSCs）were isolated and induced to differentiate into cardiomyocyte-like cells.Cardiomyocyte-like cells transfected by recombinant adenoviral vector（rAd）carrying green fluorescent protein（GFP）and human HCN4 gene were named as rAd/GFP/HCN4 group.Cardiomyocytelike cells transfected by rAd carrying GFP gene were named as rAd/GFP group.Cardiomyocyte-like cells without transfection were as controls（control group）.Western blot was used to detect the expression of HCN4 protein in each group,and whole-cell patch clamp technique was used to record the pacemaker current in each group.Results Spindleshaped cardiomyocyte-like cells were observed by fluorescence microscopy.PCR showed that rAd carrying GFP and HCN4 gene were successfully constructed.The relative expression of HCN4 protein in rAd/GFP/HCN4 group（1.31±0.02）was significantly higher than that in rAd/GFP group（0.55±0.02）（P0.05）and control group（0.52±0.03）（P〈0.05）,and there was no significant difference between rAd/GFP group and control group（P〉0.05）.Pacemaker current was found in rAd/GFP/HCN4 group,but was not found in rAd/GFP group and control group by patch clamp technique.Conclusion HCN4 protein is highly expressed and the pacemaker current is found in cardiomyocyte-like cells transfected by rAd/GFP/HCN4,resulting in autonomous pulsation of cardiomyocyte-like cells,which might provide a research basis for creating biological pacemakers in vitro.

关 键 词：心肌样细胞 超级化激活环核苷酸门控阳离子通道4基因 骨髓间充质干细胞 起搏电流:大鼠 

分 类 号：R541.7[医药卫生—心血管疾病]