ISL-1和Tbx18联合转染对乳鼠心室肌细胞重编程的作用研究  

The effect of combined transfection of ISL-1 and Tbx18 on reprogramming of neonatal rat ventricular cardiomyocytes

作　　者：张健 杨安康 杨媚[1] 唐艳红[1] 王晞[1] 赵庆彦[1] 王腾[1] 陈玉婷[1] 黄从新[1] ZHANG Jian;YANG Ankang;YANG Mei;TANG Yanhong;WANG Xi;ZHAO Qingyan;WANG Teng;CHEN Yuting;HUANG Congxin(Department of Cardiology,Renmin Hospital of Wuhan University,Cardiovascular Research Institute of Wuhan University,Hubei Key Laboratory of Cardiology,Hubei 430060,China)

机构地区：[1]武汉大学人民医院心内科,武汉大学心血管病研究所,心血管病湖北省重点实验室,430060

出　　处：《国际心血管病杂志》2018年第6期351-357,共7页International Journal of Cardiovascular Disease

基　　金：湖北省技术创新专项(重大项目)(2016ACA153);中央高校基本科研业务费专项资金(2042015kf0229)

摘　　要：目的:探讨ISL-1和Tbx18联合转染对乳鼠心室肌细胞重编程的作用,及能否在体外构建生物起搏点。方法:将乳鼠心室肌细胞随机分成空白对照组、GFP组、ISL-1组、Tbx18组、ISL-1+Tbx18组,分别转染相应病毒,培养5～7 d后qRT-PCR、Western blot和免疫荧光检测超极化激活环核苷酸门控离子通道蛋白亚型4(HCN4)的表达情况,观察细胞形态和搏动频率变化,并用膜片钳技术记录细胞内电流活动。结果:ISL-1组、Tbx18组和ISL-1+Tbx18组的细胞搏动频率和HCN4的mRNA和蛋白表达水平较空白对照组和GFP组明显升高,其中ISL-1+Tbx18组的细胞搏动频率和HCN4的mRNA和蛋白表达水平明显高于ISL-1组和Tbx18组(P均<0.05)。通过免疫荧光技术,ISL-1组和Tbx18组可检测到HCN4不连续表达,ISL-1+Tbx18组HCN4连续高表达。通过膜片钳技术,ISL-1组和Tbx18组均仅有少数细胞可记录到起搏电流活动,而ISL-1+Tbx18组大多数细胞可记录到起搏电流活动。结论:ISL-1和Tbx18联合表达能提高乳鼠心室肌细胞重编程为起搏样细胞的效率。Objective:To investigate the effect of combined transfection of ISL-1 and Tbx18 on reprogramming of neonatal rat ventricular cardiomyocytes and whether it could construct biological pacemaker in vitro.Methods:The neonatal rat ventricular cardiomyocytes were randomly divided into five groups:Bank,GFP,ISL-1,Tbx18,ISL-1+Tbx18,which were transfected virus by grouped.After cultured 5~7 d,we observed the changes of cell morphology and beat rate,estimated the expression of hyperpolarization-activated cyclic nucleotide-gated channels protein subtype 4(HCN4)by real-time fluorescence quantitative polymerase reaction(qRT-RCR),western blot,and immunofluorescence,and recorded cell current activity by patch clamp technique.Results:Compared with the bank group and GFP group,the cell beat rates and the expression levels of HCN4 mRNA and protein were significantly increased in ISL-1 group,Tbx18 group and ISL-1+Tbx18 group.Of those,the mRNA and protein expression level of HCN4 and the cell beat rate in ISL-1+Tbx18 group were significantly higher compared with ISL-1 group and Tbx18 group(all P<0.05).By immunofluorescence,ISL-1 group and Tbx18 group could be detected discontinuous expression of HCN4,meanwhile the ISL-1+Tbx18 group showed a continuous high expression of HCN4.The pacing current activity could be recorded by the patch clamp technique in the experimental group,while most of the cells could record the pacing current activity in group ISL-1+Tbx18.Conclusions:The combination of ISL-1 and Tbx18 could improve the efficiency of reprogramming that neonatal rat ventricular cardiomyocytes translate into pacemaker-like cells.

关 键 词：生物起搏 ISL-1 Tbx18 重编程 

分 类 号：R541.7[医药卫生—心血管疾病]