【摘要】 旨在探讨瞬时电位受体M2离子通道(TRPM2)在H9N2流感病毒感染小鼠肺微血管内皮细胞(PMVEC)导致线粒体损伤过程中的作用。在已建立TRPM2 shRNA PMVEC的基础上,采用5MOI H9N2猪流感病毒感染细胞,在病毒作用后24和48 h提取各组细胞的线粒体进行蛋白定量,检测各组细胞线粒体超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、一氧化氮合成酶(NOS)、线粒体呼吸链复合物Ⅳ活性和ATP酶水平;利用激光共聚焦显微镜观察线粒体膜电位变化(JC-1染色)及细胞凋亡(Annexin V-FITC/PI双染色法)情况。结果表明:H9N2-SIV感染后TRPM2 shRNA PMVEC内SOD活性、GSH水平和Na~+-K~+-ATP、线粒体呼吸链复合物Ⅳ活性显著高于对照shRNA PMVEC(P<0.05或P<0.01);mtNOS活性则极显著低于shRNA PMVEC(P<0.01)。JC-1染色显示TRPM2-siRNA PMVEC线粒体膜电位水平高于对照shRNA PMVEC,但是AnnexinV-FITC/PI染色显示细胞凋亡则低于H9N2感染对照shRNA PMVEC。结果提示:TRPM2基因沉默有效减缓H9N2感染导致NOS产生,显著降低SOD、GSH、线粒体呼吸链复合物Ⅳ、Na~+-K~+-ATP的消耗以及线粒体膜电位的下降程度,进而有效缓解PMVEC线粒体损伤及细胞凋亡。更多还原

【Abstract】 This study aimed to investigate the effect of transient receptor potential melastatin 2(TRPM2) ion channel on pulmonary microvascular endothelial cell(PMVEC) mitochondrial damage induced by H9N2 influenza virus infection. Basing on the established TRPM2 shRNA PMVEC, in this study, TRPM2 shRNA PMVEC and shRNA PMVEC(control) were infected by the dose of five multiplicity of infection(MOI) H9N2 influenza virus. At 24 and 48 h post-infection, the mitochondria of PMVECs mentioned above was extracted and the protein level was measured by BCA protein assay kit; The SOD activity, GSH level, NOS activity, Na~+-K~+-ATPase and mitochondrial complex Ⅳ were measured according to the manufacturer’s instructions;The mitochondrial membrane potential change(using mitochondrial membrane potential assay kit with JC-1) and apoptosis(using annexin V-FITC/PI apoptosis detection kit) of PMVEC were observed by laser scanning confocal microscope. The results showed that the activity of SOD, Na~+-K~+-ATPase and mitochondria complex Ⅳ and the level of GSH in TRPM2-shRNA PMVEC were significantly higher than those of shRNA PMVEC(control) after H9N2 influenza virus infection(P<0.05 or P<0.01); However, the NOS activity was dramatically lowered than that of control PMVEC(P<0.01). Moreover, the observation of electron microscope showed that the damage of organelles of TRPM2-shRNA PMVEC was not as serious as that of control shRNA PMVEC; Furthermore, the mitochondrial membrane potential level also was higher than that of control shRNA PMVEC. The apoptosis of TRPM2 shRNA PMVEC detected by annexin V-FITC/PI staining showed that there were less PMVECs stained by green fluorescence in cytomembrane and red fluorescence in nucleus than that of control shRNA PMVEC. The results demonstrated that gene silencing of TRPM2 by shRNA alleviated effectively SOD, GSH, Na~+-K~+-ATPase, mitochondrial complex Ⅳ in PMVEC infected by H9N2 virus, and further to prevent the mitochondrial damage and apoptosis of PMVEC.更多还原