本文選題：心肌缺血 + HCN�。� 參考：《臨床心血管病雜志》2017年12期

【摘要】：目的:觀察乳兔竇房結(jié)細(xì)胞在缺血條件下HCN4的表達(dá)變化及其對(duì)竇房結(jié)功能及細(xì)胞凋亡的影響。方法:將原代培養(yǎng)的乳兔竇房結(jié)細(xì)胞分為正常對(duì)照組和模擬單純?nèi)毖M(缺血1h、3h、6h);采用RT-PCR和Western blot法檢測(cè)細(xì)胞中HCN1-4的表達(dá);采用細(xì)胞免疫熒光方法檢測(cè)竇房結(jié)細(xì)胞內(nèi)HCN4蛋白的亞細(xì)胞分布;采用RT-qPCR檢測(cè)Bax和Bcl-2的表達(dá);采用全細(xì)胞膜片鉗記錄單個(gè)竇房結(jié)細(xì)胞的If電流,并運(yùn)用HCN通道抑制劑伊伐布雷定觀察抑制HCN4對(duì)乳兔竇房結(jié)細(xì)胞電活動(dòng)的影響。結(jié)果:原代乳兔竇房結(jié)細(xì)胞主要表達(dá)HCN4,少量表達(dá)HCN1和HCN2,HCN3 mRNA未檢出,Western blot僅檢測(cè)出HCN4,未檢測(cè)出HCN1、HCN2、HCN3的蛋白水平,HCN4蛋白廣泛分布于細(xì)胞質(zhì)內(nèi);膜片鉗可記錄到單一竇房結(jié)細(xì)胞自發(fā)起搏電流,伊伐布雷定可通過(guò)特異性的降低竇房結(jié)舒張期去極化速率來(lái)延緩自發(fā)活動(dòng),伊伐布雷定可持續(xù)抑制竇房結(jié)細(xì)胞的If電流產(chǎn)生,使If明顯減小;與正常對(duì)照組相比,隨著缺血時(shí)間的延長(zhǎng)(1h、3h、6h),HCN4mRNA表達(dá)及蛋白水平逐漸減少,同時(shí)Bax mRNA表達(dá)及蛋白水平明顯上調(diào),Bcl-2mRNA表達(dá)及蛋白水平明顯下降;急性缺血處理顯著抑制了竇房結(jié)細(xì)胞的電活動(dòng),與正常對(duì)照組相比,隨著時(shí)間的延長(zhǎng),竇房結(jié)細(xì)胞的起搏電流If也逐漸減少。結(jié)論:HCN4蛋白參與調(diào)控乳兔竇房結(jié)細(xì)胞的起搏功能。急性缺血可引起竇房結(jié)細(xì)胞凋亡,導(dǎo)致HCN4的表達(dá)下降,使得通道If電流密度減少導(dǎo)致竇房結(jié)功能障礙。
[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.
【作者單位】： 三峽大學(xué)第一臨床醫(yī)學(xué)院心血管內(nèi)科;三峽大學(xué)醫(yī)學(xué)院;
【基金】：湖北省自然科學(xué)基金(No:2015CFB287)
【分類號(hào)】：R541.7

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