HBx基因通过ERK1/2信号通路促肝细胞增殖及炎性因子表达  

HBx gene accelerates cell proliferation and enhances inflammatory factors expression through ERK1/2 signaling pathway

作　　者：黄欣 张思遥 王薛 杜彬 毋楠 周梦瑶 冯涛[1] HUANG Xin;ZHANG Siyao;WANG Xue;DU Bin;WU Nan;ZHOU Mengyao;FENG Tao(Molecular Medicine and Cancer Research Centre,Department of Biochemistry and Molecular Biology,College of Basic Medical Sciences,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学分子医学与肿瘤研究中心,基础医学院生物化学与分子生物研究中心,重庆400016

出　　处：《第三军医大学学报》2020年第8期790-798,共9页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81071770)。

摘　　要：目的利用稳定表达HBx基因的小鼠动物模型探讨HBx基因在体内对肝脏细胞周期和肝脏炎症活动的影响。方法利用携带HBx基因的慢病毒感染肝前体细胞14-19,小鼠肝门静脉注射经改造的肝前体细胞以构建表达HBx基因的小鼠模型。利用qRT-PCR、Western blot和免疫组化方法检测肝内HBx基因的mRNA和蛋白表达情况。建模后180 d检测小鼠肝组织的细胞周期调控因子CDK4和CyclinD1,肿瘤坏死因子-α(TNF-α)和白介素-10(IL-10)的mRNA和蛋白表达水平以及ERK1/2信号转导通路成员ERK1/2和MEK1/2的蛋白表达水平。HE染色观察HBx基因是否引起肝组织病理变化。结果稳定表达HBx基因的小鼠模型构建成功。与生理盐水组和EGFP-14-19组相比,小鼠肝脏组织在转入HBx 180d后HBx显著上调CDK4,CyclinD1,TNF-α的mRNA表达水平(P<0.01),同时显著上调CDK4,CyclinD1,TNF-α蛋白水平;HBx显著下调IL-10的mRNA和蛋白表达水平(P<0.05)。HBx显著激活ERK1/2信号转导通路。给予ERK1/2信号通路抑制剂U0126后,与对照组相比,实验组CDK4和CyclinD1,TNF-α的mRNA表达水平显著下调(P<0.05);同时,CDK4和CyclinD1,TNF-α蛋白水平显著下调,IL-10的mRNA和蛋白表达水平显著上调(P<0.01)。HE染色结果显示小鼠肝脏组织在转入HBx 180d后肝脏组织明显病变。结论HBx通过激活ERK1/2信号通路促进肝细胞增殖,与肝脏炎症有关。Objective To investigate the effects of HBx gene on hepatocyte cycle and immune system in vivo based on self-developed mouse model with stable expression of HBx gene.Methods Lentivirus carrying HBx gene were used to infect liver precursor cells 14-19.Then the modified liver precursor cells were injected into the portal vein of mice to construct the model.The model was identified with qRT-PCR,Western blot assay and immunohistochemistry.In 180 d after the model establishment,the mRNA and protein levels of cell cycle regulators CDK4 and Cyclin D1,TNF-αand IL-10 in the liver tissue were detected.While the protein levels of the proteins in the ERK1/2 and MEK1/2 signalling pathways were also measured.HE staining was used to observe the pathological changes in the liver tissue.Results The mouse model with stable expression of HBx gene was successfully constructed.Compared with the mice of normal saline group and the EGFP-14-19 group,in the mice in 180 d after the model establishment,HBx significantly up-regulated the mRNA(P<0.01)and protein levels of CDK4,Cyclin D1 and TNF-α,but down-regulated the expression of IL-10 at both levels(P<0.05).HBx significantly activated ERK1/2 signalling pathway.After the administration of ERK1/2 signal pathway inhibitor U0126,the mRNA and protein expression of CDK4,Cyclin D1,and TNF-αwas significantly down-regulated in the experimental group than the control group(P<0.05),but the mRNA and protein levels of IL-10 were in an opposite trend(P<0.01).HE staining showed that the liver tissue had obvious pathological changes in the mouse model.Conclusion HBx accelerates cell proliferation and induces liver inflammation by activating ERK1/2 signaling pathway.

关 键 词：HBX ERK1/2信号通路 细胞周期 炎症 动物模型 

分 类 号：R373.21[医药卫生—病原生物学] R730.23[医药卫生—基础医学]