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目的:研究HBV X基因与线粒体COXⅢ基因相互作用以及对线粒体功能的影响。方法:将HBV X基因真核表达载体pCDNA3-X及空载体pCDNA3转染成人肝细胞HL-7702细胞,经过2周G418筛选,获得稳定表达的新细胞株,分别命名为HL-7702/HBx和H-7702/pcDNA3,RT-PCR和Western blot方法证实HBV X基因在HL-7702/HBx细胞内的稳定表达。抽提细胞总RNA进行半定量RT-PCR及分离细胞线粒体进行Western blot检测COXⅢ表达水平变化,并通过酶动力学方法检测线粒体细胞色素氧化酶活性。结果:重组质粒pcDNA3-X转染后经G418筛选,RT-PCR和Western blot分析结果显示HL-7702-HBx细胞能够稳定表达HBV X基因。RT-PCR和Western blot结果发现HBV X基因下调COXⅢ蛋白表达而不影响mRNA水平表达。酶动力学检测发现线粒体细胞色素C氧化酶活性降低。结论:HBV X基因通过转录后水平调节COXⅢ表达,HBx通过与COXⅢ相互作用抑制线粒体细胞色素氧化酶活性。
Objective: To investigate the interaction between HBV X gene and mitochondrial COX Ⅲ gene and its effect on mitochondrial function. Methods: The eukaryotic expression vector pCDNA3-X of HBV X gene and empty vector pCDNA3 were transfected into human hepatocyte HL-7702 cells. After two weeks of G418 selection, stable cell lines were obtained and named HL-7702 / HBx and H-7702 / pcDNA3, RT-PCR and Western blot confirmed the stable expression of HBV X gene in HL-7702 / HBx cells. Total RNA was extracted for semi-quantitative RT-PCR and cell mitochondria were separated for Western blot detection of COX Ⅲ expression levels, and mitochondrial cytochrome oxidase activity was detected by enzymatic kinetic method. Results: The recombinant plasmid pcDNA3-X was transfected into HL-7702-HBx cells by G418. The results of RT-PCR and Western blot showed that the HBV X gene was stably expressed in HL-7702-HBx cells. RT-PCR and Western blot showed that HBV X gene downregulated COX III protein expression without affecting mRNA expression. Enzymatic kinetics detected mitochondrial cytochrome C oxidase activity decreased. Conclusion: HBV X gene regulates COX Ⅲ expression through posttranscriptional level, and HBx inhibits mitochondrial cytochrome oxidase activity through interaction with COX Ⅲ.