HBx通过Notch信号通路在正常免疫小鼠体内导致肝癌的作用机制  

Mechanism of hepatitis B virus X inducing hepatocellular carcinoma through Notch signaling pathway in a normal immune mouse model

作　　者：周梦瑶 杜彬 毋楠 黄欣 张思遥 况钦 吴勇 冯涛[1] ZHOU Mengyao;DU Bin;WU Nan;HUANG Xin;ZHANG Siyao;KUANG Qin;WU Yong;FENG Tao(Molecular Medicine and Cancer Research Centre,Center for 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》2021年第10期883-891,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：目的构建长期携带乙型肝炎病毒X(hepatitis B virus X,HBx)的正常免疫小鼠模型,以探讨HBx在体内导致肝癌发生的机制。方法将转染HBx的肝前体细胞经肝门静脉注射入昆明小鼠体内构建动物模型,分别于术后30、90、180、360 d收取肝脏组织,Western blot和RT-qPCR检测HBx的表达,HE染色观察组织病理切片,Western blot和RT-qPCR检测Notch1、Notch4、Hes1的表达,然后于180 d腹腔注射抑制剂DAPT,Western blot和RT-qPCR检测肝脏细胞凋亡及细胞周期相关因子的表达变化。结果动物模型构建成功且于360 d发生肝癌,与对照组相比,Notch1、Notch4、Hes1的基因及蛋白水平均于30 d呈现显著上调且一直持续至360 d(P<0.05、P<0.05、P<0.001;P<0.05、P<0.05、P<0.01);Bcl-2、CDK4、CyclinE、CyclinD1的蛋白水平在HBx组上调(P<0.001;P<0.01;P<0.001;P<0.01),Bax呈下调(P<0.001),基因水平与其一致(P<0.05;P<0.001;P<0.001;P<0.01;P<0.001),而抑制剂DAPT组显著扭转上述趋势。结论HBx在体内通过持续激活Notch通路引起细胞凋亡及细胞周期异常变化,最终导致肝癌发生。Objective To construct a normal immune mouse model carrying hepatitis B virus X(HBx)for long term so as to explore the possible mechanism of HBx causing hepatocellular carcinoma(HCC).Methods Hepatic progenitor cells transfected with HBx were injected into Kunming mice via hepatic portal vein to construct the animal model.The liver tissues were harvested at 30,90,180 and 360 d after injection,respectively.The expression of HBx as well as that of Notch1,Notch4 and Hes1 at protein and mRNA levels was detected by Western blotting and RT-qPCR,respectively,and the histopathological changes were observed by HE staining.In addition,a novelγ-secretase inhibitor,DAPT,was intraperitoneally injected at 180 d,and then Western blotting and RT-qPCR were adopted to determine the changes in the expression of apoptosis and cell-cycle-related factors.Results The animal model was successfully constructed,and liver cancer occurred at 360 d.Compared with the control group,the mRNA and protein levels of Notch1,Notch4 and Hes1 were all significantly increased in the HBx group at 30 d(P<0.05,P<0.05,P<0.001),and continued to 360 d(P<0.05,P<0.05,P<0.01);the protein and mRNA levels of Bcl-2,CDK4,CyclinE and CyclinD1 were also up-regulated(protein:P<0.001,P<0.01,P<0.001,P<0.01;mRNA:P<0.05,P<0.001,P<0.001,P<0.01),while those of Bax down-regulated in the HBx group(P<0.001;P<0.001).However,the inhibitor DAPT significantly reversed the above trends.Conclusion HBx causes anti-apoptosis and abnormal cell cycle changes in mice through persistent activation of the Notch pathway,and then ultimately leads to HCC.

关 键 词：肝细胞癌 乙型肝炎病毒X 小鼠模型 NOTCH信号通路 DAPT 

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