A549细胞针对A型流感病毒的抗病毒反应  

Antiviral response of A549 cells against Influenza A virus

作　　者：杨宣叶 高明阳 胡欣妍 王进千 马晓霞[1,2] YANG Xuanye;GAO Mingyang;HU Xinyan;WANG Jinqian;MA Xiaoxia(Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;College of Life Science and Engineering,Northwest Minzu University)

机构地区：[1]西北民族大学生物医学研究中心,生物工程与技术国家民委重点实验室,甘肃兰州730030 [2]西北民族大学生命科学与工程学院

出　　处：《中国病原生物学杂志》2024年第8期880-885,共6页Journal of Pathogen Biology

基　　金：中央高校基本科研业务费专项资金资助(NO.31920220134);甘肃省自然科学基金(20JR5RA505)。

摘　　要：目的 了解IAV在A549细胞中的增殖,探究IAV感染A549细胞后所被激活的抗病毒反应。方法 以IAV感染人肺腺癌上皮细胞(A549)细胞为模型,通过实时荧光定量PCR法(qRT-PCR)、Western blot以及噬斑实验测定IAV在A549细胞中的增殖。Western blot检测IAV感染A549细胞36 h后,p-NF-κB、IκBα和p-IκBα蛋白表达水平。通过qRT-PCR检测IFN信号通路的激活以及IFN刺激基因的表达。收集IAV感染A549细胞36 h及48 h上清,与新鲜培养基混合培养IAV感染A549细胞,qRT-PCR法检测内源性IFN对IAV增殖的抑制作用。结果 IAV感染A549细胞36 h后RNA水平达到最高值4.86×10~6,Western blot与噬斑试验均证明IAV在A549细胞中有效增殖。IAV显著促进A549细胞中磷酸化NF-κBp65和磷酸化IκBα的蛋白表达。IAV可有效激活A549细胞中的IFN信号通路,并诱导产生Ⅰ型IFN(IFNβ)以及Ⅲ型IFN(IFNλ1、IFNλ2、IFNλ3)为主的细胞因子,其中36 h分别是0 h的(16.77±0.6614)、(323.5±41.88)、(3632±240.2)和(4690±231.3)倍。同时,IAV感染A549细胞后可诱导产生一系列具有广谱抗病毒作用的IFN刺激基因(IFN-stimulated genes, ISGs),如CXCL10、RIG-I、MX1、CCL5、IFI27、STAT1、ISG15,分别是对照组的(684.8±50.37)、(70.19±2.917)、(290.8±10.71)、(203.8±4.994)、(205.0±6.046)、(5.974±0.1550)和(603.0±70.25)倍。此外,收集的病毒感染36h和48 h的细胞培养上清对病毒复制均有显著的抑制作用,分别为对照组的(-0.1231±0.05704)和(-0.3519±0.05257)倍。结论 IAV感染A549细胞激活IFN与ISGs,有效促进磷酸化NF-κBp65的蛋白表达,为研究不同细胞系针对不同亚型流感病毒的抗病毒反应的差异提供参考依据,有助于进一步探究IAV与常用细胞系A549的相互作用机理。Objective To verify the proliferation of influenza A virus in A549 cells and explore the antiviral response activated by IAV infection in A549 cells. Methods Using human lung adenocarcinoma epithelial cells(A549) as a model for IAV infection, IAV proliferation in A549 cells was determined by Quantitative Real-time PCR(qRT-PCR),Western blot and Plaque assay. NF-κB,IκBα and p-IκBα protein level were detected by Western blot while the activation of IFN signaling pathway and the expression of IFN stimulated genes(ISGs) were tested through qRT-PCR. The supernatants of A549 cells infected with IAV for 36h or 48h were collected and mixed with fresh culture medium, which in turn used to culture new IAV infected A549 cells. Finally, qRT-PCR was used to detect the inhibitory effect of endogenous IFN on IAV proliferation. Results After 36 hours of IAV infection in A549 cells, the RNA level reached its highest value of 4.86×10~6.IAV proliferates efficiently in A549 cells and induces p-NF-κBp65 and p-IκBα protein expression significantly. IAV effectively activate the IFN signaling pathway in A549 cells and induce the production of cytokines dominated type Ⅰ IFN(IFNβ) and type Ⅲ IFN(IFNλ1,IFNλ2,IFNλ3) with 36 hours being(16.77±0.6614),(323.5±41.88),(3632±240.2) and(4690±231.3) times higher than 0 hours. Meanwhile, IAV infection of A549 cells can induce the production of a series of IFN stimulated genes(ISGs) with broad-spectrum antiviral effect, such as CXCL10,RIG-I,MX1,CCL5,IFI27,STAT1 and ISG15,which are(684.8±50.37),(70.19±2.917),(290.8±10.71),(203.8±4.994),(205.0±6.046),(5.974±0.1550) and(603.0±70.25) times higher than the control group. Furthermore, the cell culture supernatants collected after 36 and 48 hours of IAV infection showed significant inhibitory effects on IAV replication, which were(-0.1231±0.05704) and(-0.3519±0.05257) times higher than the control group. Conclusion In this study, IAV infection activates IFN and ISGs and significantly promoted phosphorylation of NF-κBp65proteini

关 键 词：甲型流感病毒 干扰素 NF-ΚB 抗病毒反应 

分 类 号：R392[医药卫生—免疫学]