基因1b型不同准种株丙型肝炎病毒核心蛋白对HepG2细胞生物学行为的影响  

Effects of core proteins of different quasispecies of genotype 1b hepatitis C virus on biological behaviors of HepG2 cells in vitro

作　　者：陈卫兵 王小红 刘芳[1] Chen Weibing;Wang Xiaohong;Liu Fang(Department of Infectious Diseases,First People's Hospital,Yangtze University,Xiantao 433000,Hubei Province,China)

机构地区：[1]湖北省仙桃市长江大学附属仙桃市第一人民医院感染病科,433000 [2]陆军军医大学西南医院感染病科

出　　处：《实用肝脏病杂志》2022年第1期13-17,共5页Journal of Practical Hepatology

基　　金：湖北省科技厅科研基金资助项目(编号:2019HB141)。

摘　　要：目的探讨基因1b型不同准种株丙型肝炎病毒(HCV)核心蛋白(core)对HepG2细胞生物学行为的影响。方法构建基因1b型HCV癌中心株(T)、癌旁株(NT)和C191(HCV-J6)的core重组真核表达质粒,通过Lipofe ctamine 2000转染至HepG2细胞,分别称为pcEGFP-T组、pcEGFP-NT组和pcEGFP-C191组,设置对照组和空质粒组,采用平板克隆法检测细胞增殖,采用qRT-PCR法检测细胞增殖相关基因(PCNA、Ki67、Cyclin B、CDK1)mNRA相对水平。给予细胞50 ng/mL肿瘤坏死因子-α(TNF-α)作用8 h,使用流式细胞仪检测细胞凋亡,采用Western blot法检测细胞凋亡相关蛋白(Bax/Bcl-2、Caspase-3、Caspase-9)和侵袭相关蛋白(MMP-3、MMP-9、Snail)表达情况。结果pcEGFP-NT组、pcEGFP-T组和pcEGFP-C191组HepG2细胞克隆形成率分别为(57.3±4.2)%、(64.5±3.8)%和(49.8±3.2)%,显著高于空质粒组【(44.3±3.4)%,P<0.05】,细胞凋亡显著弱于空质粒组;pcEGFP-NT组PCNA、Ki67、Cyclin B和CDK1 mNRA相对水平分别为(1.5±0.0)、(1.7±0.1)、(1.6±0.0)和(1.8±0.1),显著高于空质粒组【分别为(1.0±0.1)、(1.0±0.1)、(1.0±0.1)和(1.0±0.1),P<0.05】;pcEGFP-T组PCNA、Ki67、Cyclin B和CDK1 mNRA相对水平分别为(1.9±0.1)、(2.1±0.1)、(2.3±0.1)和(2.6±0.1),显著高于空质粒组(P<0.05),pcEGFP-C191组分别为(1.2±0.1)、(1.4±0.1)、(1.4±0.0)和(1.5±0.0),也显著高于空质粒组(P<0.05);pcEGFP-NT组、pcEGFP-T组、pcEGFP-C191组Bax/Bcl-2、Caspase-3、Caspase-9蛋白表达量显著弱于空质粒组,而MMP-3、MMP-9和Snai蛋白表达量显著强于空质粒组。结论HCV core蛋白表达量增强可提高HepG2细胞增殖和抗凋亡能力,具有重要的研究意义。Objective This experiments aimed to explore the effects of core proteins of different quasispecies of genotype 1b hepatitis C virus(HCV)on the biological behaviors of HepG2 cells in vitro.Methods The eukaryotic plasmids of genotype 1b HCV center strain(T),para-carcinoma strain(NT)and C191 strain(HCV-J6)were constructed,and the HepG2 cells were transfected by them,e.g.,pcEGFP-T,pcEGFP-NT and pcEGFP-C191 group by Lipofectamine 2000.The cells proliferation was detected by plate clone method,and the relative levels of cell proliferation related genes,such as PCNA,Ki67,Cyclin B and CDK1 mNRA were detected by fluorescent quantitative PCR.The cells were treated with tumor necrosis factor-α(TNF-α)at dose of 50 ng/mL for 8 h.The apoptosis was detected by flow cytometry.The expressions of apoptosis-related proteins Bax/Bcl-2,Caspase-3 and Caspase-9 and invasion-related proteins of MMP-3,MMP-9 and Snail were detected by Western blot.Results The clone formation rates in pcEGFP-NT-,pcEGFP-T-and pcEGFP-C191-transfected HepG2 cells were(57.3±4.2)%,(64.5±3.8)%and(49.8±3.2)%,significantly higher than[(44.3±3.4)%,P<0.05]in empty plasmid-transfected cells,while the apoptosis was significantly weaker than that in empty plasmid group;the relative levels of PCNA,Ki67,Cyclin B and CDK1 mNRA in pcEGFP-NT group were(1.5±0.0),(1.7±0.1),(1.6±0.0)and(1.8±0.1),significantly higher than[(1.0±0.1),(1.0±0.1),(1.0±0.1)and(1.0±0.1),respectively,P<0.05]in empty plasmid group,those in pcEGFP-T group were(1.9±0.1),(2.1±0.1),(2.3±0.1)and(2.6±0.1),significantly higher than in empty plasmid group(P<0.05)and those in pcEGFP-C191 group were(1.2±0.1),(1.4±0.1),(1.4±0.0)and(1.5±0.0),significantly higher than those in empty plasmid group(P<0.05);the expression levels of Bax/Bcl-2,Caspase-3 and Caspase-9 in pcEGFP-NT group,pcEGFP-T group and pcEGFP-C191 group were significantly weaker than those in empty plasmid group,while the expression levels of MMP-3,MMP-9 and Snai proteins were significantly stronger than those in empty plasmid gro

关 键 词：HEPG2细胞 丙型肝炎病毒 核心蛋白 细胞增殖 细胞凋亡 体外 

分 类 号：R512.63[医药卫生—内科学]