乙肝病毒X基因突变的人肝癌细胞稳转株的构建及其生物学行为变化  

Construction of the human hepatocellular carcinoma cell line stably expressed mutated hepatitis B virus X genes and changes of its biological behavior

作　　者：张翠芳 翟悦怡 隗和儒 李伟 王雪君 周辉芳 ZHANG Cuifang;ZHAI Yueyi;WEI Heru;LI Wei;WANG Xuejun;ZHOU Huifang(The Pingshan County People's Hospital,Shijiazhuang,Heibei 050400,China;The Third Hospital of Hebei Medical University,Shijiazhuang,Heibei 050051,China;Hebei Key Lab of Laboratory Animal Science,Hebei Medical University,Shijiazhuang,Heibei 050011,China;The 980th Hospital of PLA Joint Logistic Support Force Shijiazhuang,Heibei 050082,China)

机构地区：[1]河北省石家庄市平山县人民医院,河北石家庄050400 [2]河北医科大学第三医院,河北石家庄050051 [3]河北医科大学,河北省重点实验动物中心,河北石家庄050011 [4]中国人民解放军联勤保障部队第九八〇医院,河北石家庄050082

出　　处：《中国热带医学》2023年第10期1037-1042,共6页China Tropical Medicine

基　　金：河北省自然科学基金项目(No.C2020206052);河北省卫生健康委医学科学研究课题计划项目(No.20211094)。

摘　　要：目的构建稳定表达乙肝病毒X基因(HBx)突变(C1653T、T1753C)的HepG2、Huh7细胞株,探讨突变点对肝癌细胞生物学行为的影响。方法以含HBx的pLVX-HBx-IRES-tdTomato质粒为模板,点突变合成pLVXHBxC1653T-IRES-tdTomato、pLVX-HBxT1753C-IRES-tdTomato慢病毒质粒。双酶切和Sanger测序的方法鉴定重组质粒的准确性。空白HepG2、Huh7细胞为对照组,pLVX-HBx-IRES-tdTomato、pLVX-HBxC1653T-IRES-tdTomato、pLVX-HBxT1753C-IRES-tdTomato慢病毒液感染HepG2、Huh7细胞,0.6μg/mL嘌呤霉素筛选单克隆细胞进行扩大培养。免疫染色和Western blot法检测X蛋白(HBX)的表达鉴定稳转株的构建;CCK-8法测定细胞的增殖能力;Western blot法检测细胞中EMT相关信号分子的表达情况。两组间进行独立样本t检验。结果双酶切和Sanger测序结果显示重组慢病毒质粒酶切后片段大小正确,点突变位置及碱基替换正确,成功构建pLVX-HBx-IRES-tdTomato、pLVXHBxC1653T-IRES-tdTomato、pLVX-HBxT1753C-IRES-tdTomato慢病毒质粒。免疫染色及Western Blot结果显示转染HBx及突变型的HepG2、Huh7细胞均有HBX表达,空白对照组均无HBX表达,成功获得稳定表达HBx、HBxC1653T和HBxT1753C的HepG2、Huh7细胞稳转株。增殖实验表明HBx及突变型均较对照组明显促进肝癌细胞的增殖(P<0.01),而HBxC1653T突变较HBx进一步促进肝癌细胞的增殖(P<0.05)。同时HBxC1653T突变明显上调N-cadherin的表达、下调E-cadherin的表达,促进了上皮间质转化(epithelial to mesenchymal transition,EMT)的发生。结论成功获得稳定表达HBx、HBxC1653T和HBxT1753C的人肝癌细胞HepG2、Huh7细胞稳转株;HBxC1653T突变可导致肝癌细胞的增殖能力增强及EMT发生。Objective To construct HepG2,Huh7 cell lines stably express hepatitis B virus X(HBx)mutant(C1653T,T1753C),and explore their effect on the biological behavior of hepatocellular carcinoma cells.Methods The lentivirus plasmid of pLVX-HBxC1653T-IRES-tdTomato,pLVX-HBxT1753C-IRES-tdTomato were obtained by PCR site mutagenesis according to wild type ayr HBx.Double enzyme digestion and Sanger sequencing were performed for accuracy of plasmid.Blank HepG2 and Huh7 cells were used as the control group,HepG2,Huh7 cells were infected by pLVX-HBx-IREStdTomato,pLVX-HBxC1653T-IRES-tdTomato,and pLVX-HBxT1753C-IRES-tdTomato lentivirus solution,then monoclonal cell was selected by 0.6μg/mL puromycin.Immunostaining and Western Blot were performed for the verification of stable strains.CCK8 assay was performed for the proliferation capacity of stable strains.Western Blot was performed for expression of EMT-related signal molecules in cells.The independent samples t-test was used for comparison between two groups.Results Double enzyme digestion and Sanger sequencing showed that that the size of the cut fragments of recombinant lentiviral plasmids was correct,and the point mutation location and base substitution were correct,suggesting that the plasmid of pLVX-HBx-IRES-tdTomato,pLVX-HBxC1653T-IRES-tdTomato,pLVX-HBxT1753C-IRES-tdTomato were constructed successfully.Immunostaining and Western blot showed that HBX were expressed in stable strains,while there was no HBX expression in the blank control group,indicating that the HepG2 and Huh7 cell lines stably expressing HBx,HBxC1653T,HBxT1753C were successfully constructed.CCK8 assay showed that the proliferation capacity of HBx and mutant were enhanced compared to the control group(P<0.01),HBx C1653T displayed further additive the effect compared to HBx(P<0.05).Moreover,HBxC1653T mutation also significantly upregulated N-cadherin expression and downregulated E-cadherin expression,thus promoting the occurrence of EMT.Conclusions HepG2 and Huh7 cell lines stably expressing HBx,HBxC1653T,HB

关 键 词：乙肝病毒X基因 C1653T突变 稳转株 增殖 上皮间质转化 

分 类 号：R373.21[医药卫生—病原生物学]