神经前体细胞表达发育调控样基因4在乙型肝炎病毒复制中的作用  

Role of neural precursor cell-expressed developmentally down-regulated gene 4-like in hepatitis B virus replication

作　　者：陈邦涛 冯旭娇 杨青青[2] 刘明社[2] 赵中夫[2] 张芸[2] Chen Bangtao;Feng Xujiao;Yang Qingqing;Liu Mingshe;Zhao Zhongfu;Zhang Yun(Department of Infectious Diseases,the First Hospital of Shanxi Medical University,Taiyuan 030001,China;Institute of Liver Diseases,Changzhi Medical College,Changzhi City 046000,Shanxi Province,China)

机构地区：[1]山西医科大学第一医院感染病科,太原030001 [2]长治医学院肝病研究所,山西省046000

出　　处：《中华传染病杂志》2020年第8期501-506,共6页Chinese Journal of Infectious Diseases

基　　金：山西省高校科技创新研究项目(2016175);山西省青年科技研究基金(201601D202095)。

摘　　要：目的研究神经前体细胞表达发育调控样基因4(neural precursor cell-expressed developmentally down-regulated gene 4-like,NEDD4L)对乙型肝炎病毒(hepatitis B virus,HBV)复制的影响及其可能的分子机制。方法采用Lipofectamine2000分别将靶向NEDD4L的小干扰RNA、NEDD4L的过表达质粒(pcDNA3.1-NEDD4L-HA)、HBV复制质粒(pGEM-HBV1.3)和poly(dAT:dAT)瞬时转染至HepG2细胞,并以可稳定表达HBV的HepG2.2.15细胞为对照。采用实时荧光定量聚合酶链反应检测NEDD4L、α干扰素、β干扰素、干扰素刺激基因56(interferon-stimulated gene 56,ISG56)、黏液病毒抗性蛋白A(myxovirus resistance protein A,MxA)、寡腺苷酸合成酶(oligoadenylate synthetase,OAS)等的mRNA水平,以及HBV DNA和3.5 kb HBV RNA的表达水平;采用蛋白质印迹法验证NEDD4L沉默或过表达效果,并检测相关信号分子的蛋白质水平;采用酶联免疫吸附测定法检测细胞上清液中β干扰素产量。统计学方法采用t检验。结果HepG2.2.15细胞中的NEDD4L mRNA和蛋白质水平分别为10.53±0.47和4.17±0.43,分别高于HepG2细胞中的1.00±0.05和1.26±0.25,差异均有统计学意义(t=3.27,P=0.008;t=1.68,P=0.030)。在敲减NEDD4L基因表达的细胞中,上清液HBV DNA表达水平为0.32±0.09,低于对照组的1.00±0.05,差异有统计学意义(t=-0.93,P=0.020);3.5 kb HBV RNA表达水平为0.49±0.11,低于对照组的1.00±0.05,差异有统计学意义(t=-0.68,P=0.040)。与对照组相比,NEDD4L敲减组的β干扰素、ISG56、MxA和OAS的mRNA相对表达水平均上升,差异均有统计学意义(t=4.66、9.38、7.29、7.01,均P<0.01)。NEDD4L敲减组β干扰素含量在poly(dAT:dAT)处理和水疱性口炎病毒(vesicular stomatitis virus,VSV)刺激时分别为(776.41±115.49)ng/L和(961.21±130.19)ng/L,分别高于对照组的(320.15±56.05)ng/L和(440.17±67.82)ng/L,差异均有统计学意义(t=2.43,P=0.020;t=2.85,P=0.030);NEDD4L过表达组β干扰素含量在poly(dAT:dAT)处理和VSV刺激时分别为(156.18±26.47Objective To study the role and possible molecular mechanism of neural precursor cell-expressed developmentally down-regulated gene 4-like(NEDD4L)in the replication of hepatitis B virus(HBV).Methods Small interfering RNA(siRNA)targeting NEDD4L,plasmid expressing NEDD4L with hemagglutinin(HA)C-terminal tag(pcDNA3.1-NEDD4L-HA),plasmid expressing 1.3×HBV genome(pGEM-HBV1.3)and poly(dAT:dAT)were respectively transfected into HepG2 cells using Lipofectamine2000.HepG2.2.15 cells,a cell line that can stably express HBV,were used as control.The mRNA levels of NEDD4L,interferon(IFN)-α,IFN-β,interferon-stimulated gene 56(ISG56),myxovirus resistance protein A(MxA),oligoadenylate synthetase(OAS),and the levels of HBV DNA or 3.5 kb HBV RNA were detected by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).Western blot was used to detect the silence and over-expression of NEDD4L,and the protein levels of the related signaling molecules.The amount of IFN-βin the cellular supernatant was measured by enzyme linked immunosorbent assay(ELISA).Student t test was used for comparison of continuous data between groups.Results The levels of NEDD4L mRNA and protein in HepG2.2.15 cells were 10.53±0.47 and 4.17±0.43,respectively,which were both statistically higher than those in HepG2 cells(1.00±0.05,t=3.27,P=0.008 and 1.26±0.25,t=1.68,P=0.030,respectively).In HepG2 cells with knockdown of NEDD4L,the expression level of HBV DNA in cellular supernatant was 0.32±0.09,which was statistically lower than that in the control(1.00±0.05,t=-0.93,P=0.020),and the expression level of 3.5 kb HBV RNA was 0.49±0.11,which was statistically lower than that in the control(1.00±0.05,t=-0.68,P=0.040),while the mRNA levels of IFN-βand downstream effector molecules(ISG56,MxA and OAS)were all significantly increased compared with the control(t=4.66,9.38,7.29 and 7.01,respectively,all P<0.01).With poly(dAT:dAT)treatment and vesicular stomatitis virus(VSV)stimulation,the levels of IFN-βin HepG2 cells with knockdown of NEDD4L were

关 键 词：肝炎病毒 乙型 神经前体细胞表达发育调控样基因4 干扰素类 

分 类 号：R512.62[医药卫生—内科学]