抗HCV复制调控区药物体外筛选模型的建立

目的: 建立抗丙型肝炎病毒(HCV)复制调控区体外药物筛选模型。方法: 将全长HCV的5'非翻译区(5' UTR)的cDNA克隆至启动子探测载体pEGFP-1,构建重组载体p5' NCR-EGFP,并转染至人肝癌细胞系HepG₂。获得的转染细胞以荧光显微镜检测及原位杂交法验证,并经流式细胞术分选。通过干扰素与利巴韦林2种常规抗病毒药物对所构建的模型进行初步应用。结果: 荧光显微镜下观察到绿色荧光蛋白(EGFP)的强表达,原位杂交检测到转染细胞的核区有特异性杂交信号。流式细胞术可检测到表达EGFP的阳性细胞达95%以上。初步运用结果显示,γ干扰素对该模型有明显的抑制作用,而利巴韦林及α-2b干扰素在本模型上的抑制作用较弱。结论: 该细胞模型经验证可以成为体外筛选抗HCV复制调控区药物的模型。

Objective: To establish in vitro drug screening system for anti-hepatitis C virus (HCV)replication regulatory region. Methods: The full-length cDNA of HCV-5' untranslated region (5' UTR)was cloned into the promoter probe vector pEGFP-1 to obtain recombinant vector p5' NCR-EGFP which was then transfected into the human hepatoma cell strain HepG₂.The transfected cells obtained were verified by fluorescence microscopy assay and in situ hybridization,and screened by flow cytometry sorting (FCS).The cell-based drug screening system was preliminarily validated by using interferon and ribavirin as testing drugs. Results: Strong expression of the enhanced green fluorescent protein (EGFP)was observed under the fluorescence microscope,and specific hybridization signals in the nucleus area were detected by in situ hybridization.Percentage of positive cells expressing EGFP detected by flow cytometry (FCM)was more than 95%.The preliminary results showed that the inhibition of interferon-γ on EGFP expression in this model was significant,while the efficiencies of the ribavirin and interferon-α-2b on this model were relatively lower. Conclusion: The cell-based model could be validated as in vitro screening system of drugs for anti-HCV replication control region.