上调Cx43对心肌细胞间通讯及离子通道的影响

Changes of inter-cellular communications and cell ion channels after over-expression Cx43

目的通过重组腺病毒介导缝隙连接蛋白Cx43在体外培养的心肌细胞中过度表达,观察心肌细胞间通讯及细胞离子通道变化。方法通过RT-PCR法克隆SD大鼠缝隙连接蛋白Cx43基因的全长cDNA,用基因重组的方法构建腺病毒pAdEasy-1-Cx43。行心肌细胞基因转染后,分别用RT-PCR和蛋白质免疫印迹技术检测Cx43在心肌细胞中的mRNA和蛋白表达,用免疫荧光技术分析Cx43在心肌细胞膜上的分布。采用划痕标记荧光染料示踪(SLDT)技术观察培养心肌细胞群之间的罗氏黄(lucifer yellow,LY)染色传输,检测上调Cx43对心肌细胞间通讯的影响;采用单细胞膜片钳技术分析各离子通道电流的变化,观察上调Cx43对心肌细胞膜离子通道表达的影响。结果腺病毒重组质粒pAdEasy-1-Cx43构建成功;转染心肌细胞后,pAdEasy-1-CX43转染组Cx43mRNA及蛋白表达上调,且心肌细胞膜上分布较多;细胞划痕荧光染料示踪提示转染后,心肌细胞间通讯增强;膜片钳全细胞膜电流提示转染后钙内流降低,钾离子电流未发现有明显改变。结论成功构建腺病毒重组质粒pAdEasy-1-Cx43载体,隙连接蛋白Cx43表达上调增强了心肌细胞间通讯以及钙内流,为Cx43改善心室肌电重构,减少或预防室性心律失常发生的进一步研究打下了基础。

Objective In order to observe the changes of inter-cellular communications and cell ion channels,overexpression gap junction protein connexin 43（Cx43）was transfected in cardiomyocytes by recombinant adenovirus-mediated in vitro.Methods The full-length cDNA of Cx43 gene was cloned by RT-PCR in SD rats,and adenovirus pAdEasy-1-Cx43 was constructed through gene recombination.The expression of Cx43 mRNA was determined by RT-PCR.The protein was determined by Western blot after transfection in cardiomyocytes.The Cx43＇s distribution on myocardial cell membrane was observed by immunofluorescence.The changes of inter-cellular communication was observed by ScrapeLoading and Dye Transfer Technique（SLDT）,which was contributed to analysis lucifer yellow（LY）in cultured cardiomyocytes after over-expression Cx43;the changes of ion channel currents was observed by single-cell patch-clamp technique to analysis the expression of myocardial cell membrane ion channels after upregulation the expression of Cx43.Results The recombinant adenovirus pAdEasy-1-Cx43 was successfully constructed.Cx43 mRNA and protein expression upregulated in pAdEasy-1-CX43 group and the distribution of Cx43 was increased on myocardial cell membrane after transfection.The inter-cellular communication enhanced after transfection by Scrape-Loading and Dye Transfer Technique;whole-cell patch-clamp current calcium influx prompted after transfection.The potassium ion currents had not a similar change.Conclusion The recombinant adenovirus pAdEasy-1-Cx43 vectors were successfully constructed.The inter-cellular communication and calcium influx were enhanced after upregulation Cx43 in cardiomyocytes.These will contribute to the further study of Cx43 in improving of ventricular electrical remodeling and reducing or preventing ventricular arrhythmias.