干扰素α对丙型肝炎病毒复制子的抑制作用

Investigating the inhibitory effects of interferon-α on the replication of hepatitis C virus replicon

目的利用丙型肝炎病毒(HCV)复制子细胞模型,体外研究干扰素α(IFN-α)对HCV复制子的抑制作用,并探讨 IFN-α信号传导与激活转录分子( STAT )及介导 IFN-α抗病毒作用的干扰素刺激基因(ISGs)的表达水平.方法以HCV复制子质粒为模板体外转录合成HCV复制子RNA, 将此RNA 转染Huh7肝癌细胞并经G418筛选,建立HCV复制子细胞株;用不同剂量(0～5000 IU/ml) IFN-α处理HCV复制子细胞72 h或用1000 IU/ml IFN-α处理细胞不同时间(0、 24、 48、 72、 96h),通过半定量RT-PCR及实时定量PCR同时检测HCV RNA,Western印迹检测HCV NS5A蛋白表达水平,研究IFN-α对HCV复制子的抑制作用.结果 IFN-α能明显抑制HCV RNA的复制,10 IU/ml 及25 IU/ml IFN-α可使HCV RNA较无IFN-α处理的对照细胞分别降低约68%及75%;IFN-α 1000 IU/ml作用24 h或96 h导致HCV RNA较对照组分别降低75%及88%,同样,IFN-α对HCV NS5A蛋白的合成也有明显抑制作用,说明IFN-α的抗HCV作用呈剂量及时间依赖性;HCV复制子细胞中有抗病毒作用的 ISG(PKR、2'5'OAS、G1P3、ISG20及ISGF3γ)呈明显的IFN诱导性表达.结论 IFN-α能明显抑制HCV RNA的复制,其抑制作用与IFN-α剂量及作用时间有关,PKR、2'5'OAS、G1P3、ISG20及ISGF3γ等ISG可能介导IFN-α的抗HCV作用.

Objective To investigate the inhibitory effects of interferon-α（IFN-α） on HCV replicon and evaluate the expressing levels of signal transducer and activator of transcription genes （STAT1 and STAT2） and IFN-α stimulated genes （ ISGs ） which may mediate the inhibitory effects of IFN-α on HCV. Methods Firstly HCV replicon cell culture system was established by transfecting HCV replicon RNA transcribed in vitro into Huh？ cells and screeing with G418. Secondly, the established HCV replicon cells were treated with various concentrations of IFN-α（0, 10, 25, 50, 100, 250, 500,750, 1000, 2500, and5000 IU/ml） for72 h or treated with 1000 IU/ml of IFN-α for different lengths of time （0, 24, 48, 72, 96 h）, then the levels of HCV RNA and NS5A protein in these cells were examined by semi-quantitative RT-PCR and Western blot respectively. Results IFN-α could effectively inhibit HCV RNA replication. 10 IU/ml or 25 IU/ml of IFN-α could lead to about 68% and 75% of HCV-RNA reduction respectively. The cells treated with 1000 IU/ml IFN-α for 24 h or 96 h had about 75% and 88% of HCV RNA reduction compared with the cells of control, demonstrated that the inhibitory effects of IFN-α on HCV replicon are in dose and time dependent manners. The expressions of antiviral ISGs - PKR、2′5′OAS、G1P3、ISG20及 ISGF3γ are strongly induced by IFN-α. Conclusions HCV replicons were sensitive to IFN-α treatment. The inhibitory effects of IFN-α on HCV RNA and NS5A are both dose and time dependents. PKR、2′5′OAS、G1P3、ISG20及ISGF3γ may mediate the inhibitory effects of IFN-α on HCV replicon replication.