过表达HBx的肝细胞对肝星状细胞增殖和活化的影响及其机制  

Effect of hepatitis B x gene-overexpressed hepatocytes on the proliferation and activation of hepatic stellate cells and related mechanism

作　　者：任婷玉 聂赫中容 肖利佳 林芳楠 王大明 宋春丽 周义文 REN Tingyu;NIE Hezhongrong;XIAO Lijia;LIN Fangnan;WANG Daming;SONG Chunli;ZHOU Yiwen(Department of Clinical Laboratory,Shenzhen Hospital,Southern Medical University,Shenzhen,Guangdong 518000,China;Genetic Diagnosis Center,The First Hospital of Jilin University,Changchun 130012,China)

机构地区：[1]南方医科大学深圳医院检验科,广东深圳518000 [2]吉林大学第一医院基因诊断中心,长春130012

出　　处：《临床肝胆病杂志》2021年第7期1561-1566,共6页Journal of Clinical Hepatology

基　　金：国家自然科学基金面上项目(81974457);国家自然科学青年基金项目(81702088);广东省自然科学基金项目(2018A030313740,2018A030310446,2019A1515012079);广东省医学科学技术研究基金项目(B2018013);南方医科大学深圳医院苗苗计划项目(2018MM02);深圳市宝安区科技计划基础研究项目(2019JD443)。

摘　　要：目的探讨HBV感染对肝星状细胞(HSC)活化的影响及作用机制。方法收集2020年11月—2021年1月慢性乙型肝炎患者血浆30份、乙型肝炎肝硬化患者血浆42份、肝细胞癌患者血浆30份及健康体检者(健康对照组)的血浆18份,ELISA法检测血浆和条件培养液中HBx、TGFβ1、多巴胺β羟化酶(DBH)和羟脯氨酸(Hyp)的含量。采用LO2细胞构建过表达HBx稳转株细胞,LO2细胞分为LO2-HBx组(稳定表达HBx)、阴性对照组(LO2-con)、空白组,分别制备LO2-HBx、LO2-con和LO2细胞(Mock)的条件培养基,孵育人HSC株LX-2,分为LX-2/LO2-HBx、LX-2/LO2-con、LX-2/Mock 3组,采用CCK-8法检测各组细胞增殖变化。采用rhTGFβ1刺激LX-2细胞,另采用TGFβ1受体抑制剂处理LX-2/LO2-HBx组细胞。荧光定量PCR或Western Blot法检测LO2细胞中的HBx及上述LX-2细胞中α-SMA、Col1A1、DBH和TGFβ1的表达。多组间比较采用单因素方差分析,进一步比较方法采用Bonferroni法;2组间比较采用t检验;相关性分析采用Pearson法。结果LO2-HBx可稳定表达HBx蛋白,其培养上清中TGFβ1含量升高(F=324.701,P<0.01);共培养LX-2/LO2-HBx组细胞发生明显细胞形态变化,出现细胞收缩,胞突明显伸长,胞内脂滴减少,与LX-2/LO2-con组比较其增殖活力明显增强(P<0.05),且α-SMA和Col1A1的mRNA(F值分别为144.712和76.680,P值均<0.01)及蛋白(F值分别为234.142和528.708,P值均<0.001)表达水平升高;LX-2/LO2-HBx组细胞中TGFβ1 mRNA(F=29.382,P<0.01)及DBH mRNA水平升高(F=42.662,P<0.01)。随着rhTGFβ1刺激浓度的增加,LX-2细胞中α-SMA(F=1794.031,P<0.01)、Col1A1(F=91.340,P<0.01)及DBH(F=2501.011,P<0.01)表达增加,在rhTGFβ110 ng/ml时达到峰值。在LO2-HBx组条件培养液中加入TGFβ1受体抑制剂后LX-2细胞中DBH和Col1A1的表达较对照组下调(t值分别为3.603、5.798,P值均<0.05)。慢性乙型肝炎、乙型肝炎肝硬化、肝细胞癌患者的血浆TGFβ1(F=51.188,P<0.001)、HBx(F=39.227,P<0.001)、DBH(F=34.431,P<0.001)及HyObjective To investigate the effect of hepatitis B virus(HBV)infection on the activation of hepatic stellate cells(HSCs)and its mechanism of action.Methods A total of 30 plasma samples of chronic hepatitis B patients,42 plasma samples of hepatitis B cirrhosis patients,30 plasma samples of hepatocellular carcinoma patients,and 18 plasma samples of the individuals undergoing physical examination were collected from November 2020 to January 2021,and ELISA was used to measure the content of hepatitis B X protein(HBx),transforming growth factor-β1(TGFβ1),dopamine beta-hydroxylase(DBH),and hydroxyproline(HYP)in plasma and conditioned medium.LO2 cells were used to establish a cell line with stable overexpression of HBx(LO2-HBx)and negative control cells(LO2-con),and a conditioned medium was prepared for LO2-HBx,LO2-Con,and LO2 cells(Mock),respectively;human HSC cell line LX-2 was incubated and divided into LX-2/LO2-HBx,LX-2/LO2-con,and LX-2/Mock groups,and CCK-8 assay was used to measure the change in cell proliferation.LX-2 cells were stimulated by rhTGFβ1,and the cells in the LX-2/LO2-HBx group were treated with a TGFβ1 receptor inhibitor.Quantitative real-time PCR and Western blot were used to measure the expression of HBx in LO2 cells and the expression of alpha-smooth muscle actin(α-SMA),collagen type I alpha 1(Col1A1),DBH,and TGFβ1 in the above LX-2 cells.An analysis of variance was used for comparison between multiple groups,and the Bonferroni method was used for further comparison;the t-test was used for comparison between two groups;the Pearson method was used for correlation analysis.Results LO2-HBx stably expressed HBx protein and showed an increase in the content of TGFβ1 in supernatant(F=324.701,P<0.01).The co-cultured LX-2/LO2-HBx group had a significant change in cell morphology,with the presence of cell shrinkage,extended cytoplasmic process,and reduced lipid droplets,and compared with the LX-2/LO2-con group,the LX-2/LO2-HBx group had significant increases in proliferative activity(P<0.05)and the

关 键 词：乙型肝炎 肝硬化 肝星状细胞 HBX蛋白 

分 类 号：R512.6[医药卫生—内科学] R575.2[医药卫生—临床医学]