甲型H1N1流感病毒诱导肺泡上皮细胞低氧诱导因子-1α核转位的机制  

Mechanism of nuclear translocation of hypoxia-inducible factor-1αin influenza A(H1N1)virus infected-alveolar epithelial cells

作　　者：孟潇潇[1] 郭新坤 朱勇[1] 谢晖[1] 王瑞兰[1] Meng Xiaoxiao;Guo Xinkun;Zhu Yong;Xie Hui;Wang Ruilan(Department of Critical Care Medicine,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 201602,China;Deparment of Hepatic Oncology,Xiamen Branch,Zhongshan Hospital,Fudan University,Xiamen 361015,Fujian,China)

机构地区：[1]上海交通大学附属第一人民医院急诊危重病科,201602 [2]复旦大学附属中山医院厦门医院肝内科,361015

出　　处：《中华危重病急救医学》2020年第1期8-13,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81471891)。

摘　　要：目的探讨在肺泡上皮细胞模型中甲型H1N1流感病毒(H1N1病毒)诱导低氧诱导因子-1α(HIF-1α)核转位的分子机制。方法体外培养人肺腺癌上皮细胞(A549细胞),选取对数生长期细胞用于实验。①实验1:采用感染复数(MOI)1.0的H1N1病毒感染细胞24 h,建立H1N1病毒感染的A549细胞模型(H1N1病毒感染组);并设立空白对照组。采用蛋白质免疫印迹试验(Western Blot)检测细胞中核转运受体蛋白(Importin 4、Importin 7)表达,探讨HIF-1α核转位是否依赖Importin 4、Importin 7。②实验2:采用不同MOI(0、0.1、0.5、1.0、2.0、4.0)的H1N1病毒感染细胞24 h;采用MOI 1.0的H1N1病毒感染细胞0、3、6、12、18、24、36 h。采用实时荧光定量反转录-聚合酶链反应(RT-PCR)检测细胞中胞裂蛋白9基因变异剪接体1(SEPT9_i1)mRNA表达,探讨不同MOI和感染时间对SEPT9_i1表达的影响。③实验3:采用小干扰RNA(siRNA)转染细胞24 h抑制SEPT9_i1,建立沉默SEPT9_i1的A549细胞模型(siRNA-SEPT9_i1组);并设立空白对照组和空白载体对照组(siControl组)。3组细胞转染24 h后均给予MOI 1.0的H1N1病毒感染24 h,采用实时荧光定量RT-PCR检测细胞中SEPT9_i1 mRNA表达,以探讨siRNA对SEPT9_i1的干扰效率。④实验4:将细胞分为siControl组和siRNA-SEPT9_i1组,两组细胞转染方法同实验3。转染24 h后,两组均以MOI 1.0的H1N1病毒感染细胞,于24 h采用免疫荧光法观察细胞中HIF-1α核内外分布情况;于6、12、24、36、48 h采用实时荧光定量RT-PCR检测病毒M基因表达,以明确SEPT9_i1对HIF-1α核转位和病毒复制的影响。⑤实验5:将细胞分为空白对照组(完全培养基)、SP600125组〔100μmol/L的c-Jun氨基末端激酶(JNK)信号通路抑制剂SP600125处理细胞2 h〕、H1N1病毒感染组(MOI 1.0的H1N1病毒感染细胞24 h)和H1N1病毒+SP600125组(100μmol/L的SP600125预处理2 h后,再加入MOI 1.0的H1N1病毒感染细胞24 h)。采用实时荧光定量RT-PCR检测细胞中SEPT9_i1 mRNA�Objective To investigate the molecule mechanism of nuclear translocation of hypoxia-inducible factor-1α(HIF-1α)in influenza A(H1N1)virus infected-alveolar epithelial cells.Methods Human lung adenocarcinoma epithelial cells(A549 cells)were cultured in vitro,and cells in logarithmic growth phase were selected for experiments.①Experiment 1:the A549 cell model with H1N1 virus infection was established by using H1N1 virus infected cells with multiplicity of infection(MOI)1.0 for 24 hours(H1N1 virus infection group),and the blank control group was set up.Importin 4 and Importin 7 protein expressions were detected by Western Blot to investigate whether HIF-1αnuclear translocation depended on Importin 4 or Importin 7.②Experiment 2:the A549 cells were infected with H1N1 virus under different MOI(0,0.1,0.5,1.0,2.0,4.0)for 24 hours.Then the A549 cells were infected with H1N1 virus(MOI 1.0)for different time(0,3,6,12,18,24,36 hours).The septin 9 isoform 1(SEPT9_i1)mRNA expression was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-PCR)to investigate the effect of different MOI and infection time on the expression of SEPT9_i1.③Experiment 3:a cell model with SEPT9_i1 silencing was established by transfection of small interfering RNA(siRNA)for 24 hours(siRNA-SEPT9_i1 group),and the blank control group and blank vector control group(siControl group)were set up.Then the cells in the three groups were infected with H1N1 virus(MOI 1.0)for 24 hours after 24-hour transfection,and the SEPT9_i1 mRNA expression was detected by real-time fluorescence quantitative RT-PCR to investigate the interference efficiency of siRNA-SEPT9_i1.④Experiment 4:the cells were divided into siControl group and siRNA-SEPT9_i1 group.The transfection methods of two groups was as the same as experiment 3,and then the cells were infected with H1N1 virus(MOI 1.0)after 24-hour transfection.The distribution of HIF-1αwas detected by immunofluorescence at 24 hours after infection.The M gene expression of

关 键 词：甲型H1N1流感病毒 低氧诱导因子-1Α 核转位 

分 类 号：R511.7[医药卫生—内科学]