HCN4在缺血诱导乳兔窦房结细胞损伤中的作用

发布时间：2018-04-17 04:36

本文选题：心肌缺血 + HCN　；参考：《临床心血管病杂志》2017年12期

【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。
[Abstract]:Aim: to observe the changes of HCN4 expression in sinoatrial node cells and its effects on sinus node function and apoptosis in neonatal rabbits.Methods: primary cultured rabbit sinoatrial node cells were divided into normal control group and simulated ischemia group (1 h after ischemia for 3 h and 6 h after ischemia). The expression of HCN1-4 was detected by RT-PCR and Western blot.The subcellular distribution of HCN4 protein in sinoatrial node cells was detected by cellular immunofluorescence, the expression of Bax and Bcl-2 was detected by RT-qPCR, and the if currents of single sinoatrial node cells were recorded by whole-cell patch clamp.The effects of inhibition of HCN4 on the electrical activity of sinoatrial node cells were observed by HCN channel inhibitor Ifabradine.Results: primary neonatal rabbit sinoatrial node cells mainly expressed HCN4, a small amount of HCN1 and HCN2HCN3 mRNA were not detected, only HCN4 was detected by Western blot, and the protein level of HCN1, HCN2HCN3 was widely distributed in the cytoplasm.Patch clamp could record the spontaneous pacing current of single sinus node cells, Ifabradine could delay the spontaneous activity by specifically reducing the depolarization rate during the diastolic phase of the sinus node, and the if current production of the sinus node cells could be continuously inhibited by ivalburetine.Compared with the normal control group, the expression of HCN4 mRNA and the protein level of HCN4 decreased gradually with the prolongation of ischemia time, while the expression and protein level of Bax mRNA increased obviously. The expression of Bcl-2 mRNA and protein level decreased obviously with the prolongation of ischemia time.Acute ischemic treatment significantly inhibited the electrical activity of sinoatrial node cells. Compared with the normal control group, the pacing current if of the sinus node cells decreased gradually with the prolongation of time.ConclusionHCN4 protein is involved in the regulation of pacing function of sinoatrial node cells in neonatal rabbits.Acute ischemia can induce apoptosis of sinoatrial node cells, decrease the expression of HCN4, decrease the current density of channel if, and lead to dysfunction of sinus node function.
【作者单位】：三峡大学第一临床医学院心血管内科;三峡大学医学院;
【基金】：湖北省自然科学基金(No:2015CFB287)
【分类号】：R541.7

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