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目的 探讨腺病毒介导短发夹RNA下调Nkx2.5基因表达对体外培养大鼠乳鼠心肌细胞(NRVMs)的影响.方法 采用胰酶和Ⅱ型胶原酶混合消化法分离NRVMs,随机分为阴性对照组(NC组)和实验组.实验组转染携带靶向Nkx2.5的RNA干扰序列(短发夹RNA,shRNA)和绿色荧光蛋白(GFP)的腺病毒Ad-Nkx2.5-shRNA-GFP,NC组转染等量的仅携带GFP的对照组腺病毒Ad-GFP.以不同感染复数(MOI)转染细胞选取最适MOI,按最适MOI转染NRVMs 48h后,采用实时荧光聚合酶链式反应和蛋白质免疫印迹检测Nkx2.5沉默效果,保证沉默效果之后,检测起搏细胞发育相关转录因子(Tbx3、Tbx18、Shox2和Isl1)、起搏相关离子通道超极化激活环核苷酸门控通道4型(HCN4)和缝隙链接蛋白40(Cx40)mRNA和蛋白水平变化,采取免疫荧光法检测HCN4蛋白的表达.结果 分离培养的原代NRVMs 48h呈现成簇搏动,α-actin检测心肌纯度达0.91±0.01,转染NRVMs的最适MOI=20,构建的病毒可有效下调Nkx2.5水平(P＜0.05);下调心肌细胞Nkx2.5水平后,起搏细胞发育相关转录因子、HCN4表达水平升高(P＜0.05),心室肌相关缝隙连接蛋白Cx40表达水平下降(P＜0.05).荧光显微镜观察实验组红色荧光即离子通道HCN4表达强于对照组.结论 下调Nkx2.5可将NRVMs重编程为起搏样细胞.“,”Objective To investigate the effects of down-regulation of Nkx2.5 gene by adenovirus-mediated short hairpin RNA (shRNA) on neonatal rat cardiomyocytes (NRVMs) in vitro. Methods The NRVMs were digested and isolated by trypsin and type II collagenase.The shape of NRVMs were observed by using the opitical microscope.Immunofluorescopy was used to detect the purity of NRVMs.The cells were randomly divided into negative control group and experimental group.They were transfected with recombinant adenovirus carrying shRNA targeting Nkx2.5 and expressing green fluorescent protein (GFP), Ad-Nkx2.5-shRNA-GFP, and the control adenovirus expressing GFP only, Ad-GFP, at different multiplicities of infection (MOI) in order to select the most suitable MOI.After transfected with the selected MOI for 48 h, the expression of Nkx2.5, the levels of pacemaker-related transcription factor (Tbx18, Tbx3, Shox2, ISL1), hyperpolarization activated cyclic nucleotide gated potassium channel 4 (Hcn4), and connexin 40 (Cx40) ) mRNA and protein levels were measured by real-time PCR and Western blot.Immunofluorescopy was used to detect the expression of HCN4 channels. Results After 48 h, NRVMs isolated and cultured showed cluster-like beatings.The purity of cardiomyocytes were up to 0.91±0.01 by the detection ofα-actin.The most suitable MOI was 20.The Ad-Nkx2.5-shRNA-GFP was successfully transfected into NRVMs in vitro, and significantly down-regulated the expression of Nkx2.5 at mRNA and protein levels in the NRVMs (P ＜ 0.05);afterdown-regulation of Nkx2.5 in cardiomyocytes, the levels of pacemaker-related transcription factors and HCN4 expression were enhanced (P＜0.05);Cx40 expression was decreased (P＜0.05).Fluorescence microscopy showed that the red fluorescence of the experimental group, the expression of HCN4 channel, were stronger than that of the negative control group. Conclusion Downregulation of Nkx2.5 reprograms NRVMs into pacemaker-like cells.