丙型肝炎病毒核心蛋白激活iNOS表达对DNA的损伤作用研究

目的：在转基因小鼠中，研究丙肝病毒（HCV）核心蛋白激活诱导型一氧化氮合成酶（iNOS）表达对DNA的损伤作用。方法：携带HCVeDNA的质粒载体pSC—1／HCV显微注射入C57小鼠受精卵。取12月龄小鼠肝组织标本．RT-PCR和Western印迹检测HCV和iNOS的表达水平。尾静脉注射小分子RNA干扰片段作为实验组，PBS作为对照组。对比各组小鼠的iNOS在mRNA和蛋白水平的表达情况及其体内NO的含量。通过连接反应介导的PCR（LM-PCR）方法，评价iNOS和NO水平变化对DNA双链断裂（DSB）的影响。结果：在转基因小鼠的肝脏中，有HCV和iNOS的特异性高表达，NO含量和DSB水平也远高于未转染HCV基因的正常小鼠。经RNA干扰作用6d后，实验组小鼠的iNOS表达在mRNA和蛋白水平都有非常明显的下降（P〈0．01）；同时NO含量也下降（P〈0．01），DSB现象基本消失。结论：HCV核心蛋白在肝细胞中能激活iNOS的表达，诱导生成NO，进而引发DSB现象，造成时基因组DNA的损伤。RNA干扰可以有效逆转HCV对iNOS基因的激活，对于治疗和预防HCV患者发生的肝癌具有重要意义。

Core protein of hepatitis C virus activates expression of iNOS and enhances DNA damage

Objective: To determine the mechanisms of HCV core protein in activating the expression of iNOS and enhancing DNA damage.Methods: The vector pSG-1/HCV,containing cDNA of HCV,was micro-injected into fertilized C57 mouse eggs.The levels of HCV and iNOS in the specimens of livers from HCV transgenic mice were evaluated using RT-PCR and Western blot analysis.The small interfering RNA(siRNA) specific for iNOS was injected into the transgenic mice via caudal vein as experimental group,and PBS was injected into the controls.The expression of iNOS at mRNA and protein levels,and the production of NO were compared between these groups.The influences of iNOS and NO changes on the double-strand DNA breaks(DSB) were identified using ligation-mediated PCR.Results:There was significant overexpression of HCV and iNOS in the livers from the transgenic mice.The levels of NO and DSBs were also much higher than those in the uninfected mice.After the RNA interference,the experimental mice indicated an obvious decrease in iNOS expression at mRNA and at protein level as compared with the controls.Meanwhile,the production of NO showed an obvious decrease compared with the controls and the DSBs almost disappeared.Conclusion:HCV infection can stimulate the production of NO through activation of the gene encoding iNOS.NO will cause DNA breaks and enhance DNA mutation.RNA interference can reverse the activated expression efficiently and may be available for treatment and prevention of hepatic tumorigenesis in HCV infected patients.