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*

doi:10.3969/j.issn.1672-5069.2022.01.004

摘要/Abstract

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。

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

Abstract: 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 group. Conclusion The intensified expression of HCV core protein by special quasispecies of HCV infection might improve the proliferation and anti-apoptosis abilities of HepG2 cells in intro.

Key words: HepG2 cells, Hepatitis C virus, Core protein, Cell proliferation, Apoptosis, In vitro

陈卫兵, 王小红, 刘芳. 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^*[J]. 实用肝脏病杂志, 2022, 25(1): 13-16.

Chen Weibing, Wang Xiaohong, Liu Fang. Effects of core proteins of different quasispecies of genotype 1b hepatitis C virus on biological behaviors of HepG2 cells in vitro[J]. Journal of Practical Hepatology, 2022, 25(1): 13-16.

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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

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