HCN4在缺血诱导乳兔窦房结细胞损伤中的作用  

Role of HCN4 in ischemia induced cell injury in rabbit sinoatrial node

作　　者：滕林[1] 周飞[1] 曹春雨[2] 贺超[1] 李松[1] 丁家望[1] 杨俊[1] 

机构地区：[1]三峡大学第一临床医学院心血管内科,湖北宜昌443002 [2]三峡大学医学院

出　　处：《临床心血管病杂志》2017年第12期1218-1222,共5页Journal of Clinical Cardiology

基　　金：湖北省自然科学基金(No:2015CFB287)

摘　　要：目的:观察乳兔窦房结细胞在缺血条件下HCN4的表达变化及其对窦房结功能及细胞凋亡的影响。方法:将原代培养的乳兔窦房结细胞分为正常对照组和模拟单纯缺血组(缺血1h、3h、6h);采用RT-PCR和Western blot法检测细胞中HCN1-4的表达;采用细胞免疫荧光方法检测窦房结细胞内HCN4蛋白的亚细胞分布;采用RT-qPCR检测Bax和Bcl-2的表达;采用全细胞膜片钳记录单个窦房结细胞的If电流,并运用HCN通道抑制剂伊伐布雷定观察抑制HCN4对乳兔窦房结细胞电活动的影响。结果:原代乳兔窦房结细胞主要表达HCN4,少量表达HCN1和HCN2,HCN3 mRNA未检出,Western blot仅检测出HCN4,未检测出HCN1、HCN2、HCN3的蛋白水平,HCN4蛋白广泛分布于细胞质内;膜片钳可记录到单一窦房结细胞自发起搏电流,伊伐布雷定可通过特异性的降低窦房结舒张期去极化速率来延缓自发活动,伊伐布雷定可持续抑制窦房结细胞的If电流产生,使If明显减小;与正常对照组相比,随着缺血时间的延长(1h、3h、6h),HCN4mRNA表达及蛋白水平逐渐减少,同时Bax mRNA表达及蛋白水平明显上调,Bcl-2mRNA表达及蛋白水平明显下降;急性缺血处理显著抑制了窦房结细胞的电活动,与正常对照组相比,随着时间的延长,窦房结细胞的起搏电流If也逐渐减少。结论:HCN4蛋白参与调控乳兔窦房结细胞的起搏功能。急性缺血可引起窦房结细胞凋亡,导致HCN4的表达下降,使得通道If电流密度减少导致窦房结功能障碍。Objective：To observe the expression of HCN4 in the neonatal rabbit sinoatrial node cells under the ischemia stress and to evaluate its effect on the function and apoptosis of sinoatrial node cells.Method：The primary cultured sinoatrial node cells were divided into normal control group and ischemia group（1 h,3 h,6 h）.The expression of HCN1,2,3 and 4 were determined by RT-PCR and western-blot,and the subcellular distribution of HCN4 in sinoatrial node cells was detected by immunohistochemical straining.The expression of Bax and Bcl-2 were measured by RT-qPCR.Whole cell patch-clamp technique was used to record Ifcurrent in the sinoatrial node cell,and application of HCN channel inhibitor ivabradine to observe the effect of HCN4 on sinoatrial node cell activity.Result：The RT-PCR analysis showed that HCN4 mRNA was mainly expressed in the sinoatrial node cells,HCN1 and HCN2 mRNA were less expressed,and HCN3 mRNA was not detected in the sinoatrial node cells.Western blot analysis demonstrated that only HCN4 protein was detected and HCN1,2,3 were absent in the sinoatrial node cells.The results of immunofluorescence showed that HCN4 was widely distributed in the cytoplasm of sinoatrial node cells.The spontaneous pacemaker activity in sinoatrial node cell could be recorded by whole cell patch-clamp recording.Ivabradine could delay the spontaneous activity by slowing down the diastolic depolarization rate and it could significantly decrease Ifcurrent in sinoatrial node cell.As compared with control group,the expression of HCN4 was gradually decreased while the Bax was increased and Bcl-2 was decreased in sinoatrial node cells with the prolongation of the ischemia duration（ischemia after 1 h,3 h,6 h）.Acute ischemia significantly inhibited electric activity of sinoatrial node cells.Whole cell patch-clamp recording results showed that with the prolongation of the ischemia duration（ischemia after 1-3 h）,Ifcurrent was gradually decreased in sinoatrial node cells as compared with control group.Conclusion：These

关 键 词：心肌缺血 HCN4 窦房结细胞 细胞凋亡 

分 类 号：R363[医药卫生—病理学]