人超极化激活环核苷酸门控阳离子通道4基因体外转染乳鼠心肌细胞

Adenovirus mediated gene transfer of human hyperpolarization activated cyclic nucleotide gated channel 4 into neonatal rat cardiomyocytes.

目的观察人超极化激活环核苷酸门控阳离子通道4(hHCN4)基因对大鼠心肌细胞搏动的影响,初步探讨hHCN4转染心肌细胞构建生物起搏器的可行性。方法胰酶消化法分离培养原代乳鼠心肌细胞,随机分为实验组、空病毒对照组、空白对照组,转染时分别加入带hHCN4和绿色荧光蛋白(GFP)的腺病毒、带GFP的腺病毒、PBS,观察分析心肌细胞转染前,转染后2天和3天的搏动情况;采用逆转录聚合酶链式反应和免疫荧光检测各组心肌细胞hHCN4 mRNA和通道蛋白的表达。结果hHCN4重组腺病毒转染乳鼠心肌细胞后2天和3天较转染前搏动频率明显增加,频率高于对照组;hHCN4重组腺病毒转染乳鼠心肌细胞后,可检测到hHCN mRNA和通道蛋白表达,而对照组无表达。结论hHCN4转染心肌细胞的搏动频率增加,hHCN4基因转导入心肌细胞构建生物起搏器的方法具有初步可行性。

Objective To observe whether the spontaneous beat of neonatal rat cardiomyocytes is accelerated after in- fected with adenovirus containing human hyperpolarization activated cyclic nucleotide gated channel 4 （ hHCN4 ） and to establish a foundation for further study. Methods The neonatal rat eardiomyocytes were isolated by trypsin and were i- dentified with ct-sarcometric actin. The neonatal rat cardiomyocytes were infected with adenovirus containing hHCN4 gene. RT-PCR and immunofluoresccopy were used to detect the expression of mRNA and protein of hHCN4 channel. The beats of neonatal rat eardiomyoeytes were recorded and analysed before and after the infection. Results There were hHCN4 channel mRNA and protein expressions in rat cardiomyocytes infected with adenovirus containing hHCN4 gene, while there was no expression in control groups. The beats of neonatal rat cardiomyocytes were accelerated after infected with adenovi- rus carrying hHCN4 gene, while the controls showed no acceleration. Conclusion Transduction of hHCN4 pacemaker channel into neonatal rat cardiomyocytes can be successfully mediated by adenovirus and the spontaneous beat of cardiomyo- cytes can be accelerated with hHCN4. Overexpression of hHCN4 may be a potential method to create biological pacemaker. [ Chinese Journal of Cardiac Pacing and Electrophysiology,2009,23 （1） ：47 -50]