【摘要】 目的:探讨瞬时受体电位阳离子通道M2(TRPM2)在H9N2猪流感病毒(H9N2-SIV)感染小鼠肺微血管内皮细胞(PMVEC)损伤中的表达与作用。方法:采用H9N2-SIV感染小鼠PMVEC (每组设立3个重复),在病毒感染后24 h和48 h进行采样,根据试剂盒方法测定超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、活性氧簇(ROS)和一氧化氮合成酶(NOS)的变化;电镜观察PMVEC超微结构变化;利用Transwell培养PMVEC单层细胞,测定跨膜电阻及辣根过氧化物酶渗出率;real-time PCR和Western blot方法检测TRPM2 mRNA与蛋白的表达;采用Annexin V/PI双染法观察病毒感染细胞的凋亡情况。结果:H9N2-SIV感染后小鼠PMVEC中GSH-Px和SOD活性显著下降,而ROS水平和NOS活性显著高于未感染对照组(P<0.01);病毒感染细胞细胞器明显减少,出现空泡化结构,线粒体基质减少,细胞发生凋亡;病毒感染后跨膜电阻值明显降低,辣根过氧化物酶渗出率则显著增加;TRPM2 mRNA和蛋白表达随病毒感染时间增加显著升高(P<0.01);Annexin V/PI双染法显示病毒感染细胞的胞膜荧光显著增强,同时大量细胞的胞核显现红色,表明大量细胞发生凋亡现象。结论:TRPM2在H9N2-SIV感染PMEVC过程中表达明显增强,提示其参与了H9N2-SIV感染导致的PMVEC损伤和凋亡过程。更多还原

【Abstract】 AIM: To investigate the expression and role of transient receptor potential cation channel M2(TRPM2) in H9 N2 swine influenza virus(H9 N2-SIV) infection-induced mouse pulmonary microvascular endothelial cell(PMEVC) damage. METHODS: At 24 h and 48 h after PMEVC(3 parallel test in each group) was infected by H9 N2-SIV, the superoxide dismutase(SOD) activity, glutathione peroxidase(GSH-Px) activity, reactive oxygen species(ROS) level and nitric oxide synthase(NOS) activity were measured according to the manufacturer’s instructions. The ultrastructural changes of PMVEC were observed under electron microscope. Transendothelial electrical resistance(TEER) and the percentage of horse radish peroxidase(HRP) influx was measure by Transwell culture. The expression of TRPM2 at mRNA and protein levels was determined by real-time PCR and Western blot. The apoptosis of PMVEC was observed with Annexin V/PI staining. RESULTS: The SOD and GSH levels were decreased significantly in H9 N2-SIV-infected PMEVC as compared with control group(P<0.05). However, NOS activity and ROS level were increased dramatically(P<0.01). The organelles of PMVEC infected by H9 N2-SIV were decreased sharply, and vacuolization in mitochondria and apoptosis were also observed under electron microscope. The value of TEER was decreased significantly and the percentage of HRP influx was increased dramatically with the H9 N2-SIV infection(P<0.01). The expression of TRPM2 at mRNA and protein levels in H9 N2-SIV-infected PMVEC was increased dramatically at 24 h and 48 h as compared with control group(P<0.01). Annexin V/PI double staining showed that the cytomembrane was stained by great green fluorescence and some nuclei were stained by red fluorescence in H9 N2-SIV-infected PMVEC. However, less fluorescence stained PMVEC were found in control group. CONCLUSION: The expression of TRPM2 at mRNA and protein levels is increased significantly, which is involved in cell damage and apoptosis induced by oxidative stress of H9 N2-SIV infection.更多还原