甲型肝炎病毒细胞膜蛋白受体2促进小鼠巨噬细胞内毒素耐受形成的研究  

Hepatitis A virus cell membrane protein receptor 2 promotes endotoxin tolerance in mouse macrophages

作　　者：刘垚 刘逸 张小禾 陈睦虎 胡迎春 Liu Yao;Liu Yi;Zhang Xiaohe;Chen Muhu;Hu Yingchun(Department of Emergency Medicine,Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan,China;Department of Clinical Medicine,Southwest Medical University,Luzhou 646000,Sichuan,China)

机构地区：[1]西南医科大学附属医院急诊医学部,四川泸州646000 [2]西南医科大学临床医学院,四川泸州646000

出　　处：《中华危重病急救医学》2021年第4期472-477,共6页Chinese Critical Care Medicine

基　　金：四川省科技计划项目(2019JDPT0003)。

摘　　要：目的寻找内毒素耐受(ET)的潜在关键基因,为脓毒症的治疗提供理论和实验依据。方法①实验1(基因芯片与生物信息分析):从基因表达数据库(GEO)中下载ET相关基因数据集GSE47783,该数据集由脂多糖(LPS)刺激小鼠巨噬细胞建立脓毒症模型(LPS组)和ET模型(ET组)后进行实验获得;利用IDEP 0.92软件筛选数据集中两组差异表达基因(DEG),同时进行基因本体(GO)分析,并定位DEG主要富集的功能和信号通路。利用基因与蛋白质相互作用检索数据库(STRING),针对DEG构建蛋白质-蛋白质相互作用(PPI)网络,筛选出核心基因甲型肝炎病毒细胞膜蛋白受体2(HAVCR2)进行后续验证研究。②实验2(小鼠巨噬细胞株RAW264.7模型制备):体外培养RAW264.7细胞,通过LPS刺激制备ET模型(ET组,用10μg/L的LPS培养24 h后,再用100μg/L的LPS培养4 h)和脓毒症模型(LPS组,用100μg/L的LPS培养4 h);磷酸盐缓冲液(PBS)组给予等体积溶媒PBS培养4 h。采用实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质免疫印迹试验(Western blotting)检测细胞HAVCR2的mRNA及蛋白表达。③实验3(HAVCR2慢病毒载体转染RAW264.7细胞):为进一步明确HAVCR2是否参与ET形成,用慢病毒短发夹RNA(shRNA)技术敲低RAW264.7细胞中的HAVCR2后,再制备ET模型(HAVCR2--ET组),并设非敲低HAVCR2的对照组(ET组)。采用RT-qPCR法检测细胞中巨噬细胞极化关键基因〔精氨酸酶1(ARG1)、CD206、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、一氧化氮合酶2(NOS2)〕的mRNA表达。结果①实验1:共获得1013个DEG;与LPS组比较,ET组有521个上调基因,492个下调基因;这些DEG的功能主要为增加生物合成、抑制炎症反应,主要富集于Janus激酶/信号转导与转录激活因子(JAK/STAT)、NOD样受体、Toll样受体(TLR)、TNF、缺氧诱导因子-1(HIF-1)等信号通路。选择ET组第一个表达上调的膜蛋白HAVCR2作为验证研究的目标。②实验2:体外实验结果显示,经大Objective To screen out the potential key genes of endotoxin tolerance(ET),and to provide theoretical and experimental evidence for treatment and prognosis of sepsis.Methods①Experiment 1(gene chip and bioinformatics analysis):ET related data set GSE47783 was downloaded from the Gene Expression Omnibus(GEO).The data set was obtained from lipopolysaccharide(LPS)stimulated mouse macrophages to establish sepsis model(LPS group)and ET model(ET group).IDEP 0.92 software was used to screen differential expressed gene(DEG)between the two groups,analyze gene ontology(GO),and locate the main functions and signaling pathways of differential genes.The protein-protein interaction(PPI)network of DEG was constructed by the Search Tool for the Retrieval of Interacting Genes Database(STRING)to screen core genes hepatitis A virus cell membrane protein receptor 2(HAVCR2)for following up validation study.②Experiment 2(reproduction of mouse macrophage RAW264.7 model):RAW264.7 cells were cultured in vitro,the ET model(ET group,cells were cultured with 10μg/L LPS for 24 hours and then with 100μg/L LPS for 4 hours)and sepsis model(LPS group,cells were cultured with 100μg/L LPS for 4 hours)were reproduced by LPS stimulation.Phosphate buffer saline(PBS)group was given equal volume of solvent PBS for 4 hours.The mRNA and protein expressions of HAVCR2 were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blotting.③Experiment 3(RAW264.7 cells transfected with HAVCR2 lentiviral vector):to further clarify whether HAVCR2 was involved in the formation of ET,after knockdown of HAVCR2 in RAW264.7 cells by lentiviral short hairpin RNA(shRNA)technology,the ET model(HAVCR2--ET group)was constructed again,and the control group(ET group)without knockdown of HAVCR2 was set up.RT-qPCR method was used to detect the mRNA expressions of macrophage polarization key proteins[arginase 1(ARG1),CD206,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),nitric oxide synthase 2(NOS2)]in cells.Results①Expe

关 键 词：甲型肝炎病毒细胞膜蛋白受体2 内毒素耐受 脓毒症 生物信息学 

分 类 号：R459.7[医药卫生—急诊医学]