【摘要】 目的研究铁对HCV IRES依赖的病毒蛋白翻译的影响,以及其与细胞ROS活性变化的关系。方法以脂质体细胞转染法,将双荧光素酶报告基因表达载体p CI-Rluc-HCV IRES-Fluc转染人肝癌细胞系Huh-7细胞。在0、50和300μmol/L浓度的柠檬酸铁铵(FAC)作用24 h后,双荧光素酶报告基因检测系统检测HCV IRES依赖的病毒蛋白翻译的变化。ROS荧光染色方法检测细胞ROS活性变化,Western blot法检测细胞中Nrf2蛋白表达的变化。加入100μmol/L的DPI后,检测300μmol/L FAC实验组中HCV蛋白翻译的变化和细胞ROS的变化。结果与对照组相比,FAC增加了Huh-7细胞HCV IRES依赖的病毒蛋白翻译、增加了ROS活性,并且可以引起核转录因子Nrf2表达增加(P<0.05)。加入ROS抑制剂DPI后,可以显著抑制FAC诱导的HCV IRES依赖的病毒蛋白翻译及Nrf2蛋白的表达(P<0.05)。结论 FAC可以诱导细胞HCV IRES依赖的病毒蛋白翻译增加,可能与FAC促进Huh-7细胞产生过多的ROS有关。更多还原

【Abstract】 Objective To identify the activity of HCV IRES translation differences and identify the relationship between HCV IRES translation activity and ROS in different concentrations of ferric ammonium citrate( FAC) induction. Methods 1) Expression plasmid p CI-Rluc-HCV IRES-Fluc was confirmed by endonuclease digestion as well as luciferase transient expression in Huh-7 cell; 2) Controlled by dual-luciferase reporter assay,the different translation activity of HCV internal ribosomal entry site( IRES) was examined in a concentration of50 μmol / L and 300 μmol / L of FAC induction; ROS fluorescent staining method was used to detect the activity of ROS in Huh-7 cells,Western blot method was used to detect the protein expression changes of Nrf2 in Huh-7cells; 3) On the basis of the above experiments,100 μmol / L DPI was added in 300 μmol / L FAC experimentalgroup,to analyse the changes of HCV replication and ROS production after joining DPI. Results The generation of ROS and the activity of luciferase in the model group were significantly higher than that in the control group( P < 0. 05). FAC can enhance the expression of HCV IRES and increase the production of ROS,then causing Nrf2 expression in Huh-7 cell. However,after adding ROS inhibitor DPI,the above functions in Huh-7 cell were weakened. Conclusions The increase of HCV IRES expression induced by FAC is related to excessive ROS production induced by FAC in Huh-7 cells.更多还原