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目的建立接近体内自然感染状态,并能长期稳定复制的丙型肝炎病毒(HCV)感染细胞模型。方法在含10％小牛血清的RPMI1 640培养基、37℃、5％ CO_2条件下于培养瓶中培养人肝癌细胞株HepG_2、SMMC 7721及胎肝细胞株L02细胞,L02培养基加胰岛素0.2μ/ml,用HCV阳性血清感染HepG_2、SMMC 7721、L02细胞,3株细胞均每隔3d换液,消化、传代1次,连续培养60d,用套式逆转录-聚合酶链反应(nested RT-PCR)检测培养细胞及上清液中正、负链HCV RNA。结果HepG_2、SMMC 7721细胞在感染后2～30d,L02细胞在感染后3～30d于细胞中可以间断检出HCV正链RNA;3株细胞的细胞内HCV负链RNA均在感染后3～30d可以间断检出,检出率与正链RNA接近。3株细胞以HepG_2细胞中HCV RNA正、负链检出率较高,并在第31～60天仍可间断检出HCV RNA正、负链,但复制程度逐渐减弱。HepG_2、SMMC 7721及L02 3株细胞的培养上清液中HCV正链RNA感染后也呈间断阳性,检出率与细胞内正链RNA基本一致。培养上清液中均未检出HCV负链RNA。结论 HepG_2、SMMC 7721及L02细胞均对HCV易感,并能支持HCV长期稳定的复制。因此HepG_2细胞是较为理想的HCV体外感染细胞模型。
Objective To establish a cell model of Hepatitis C virus (HCV) infection that is close to the natural infection in vivo and can be stably replicated for a long time. Methods Human hepatocellular carcinoma cell lines HepG_2, SMMC 7721 and fetal liver cell line L02 were cultured in RPMI 1 640 medium supplemented with 10% calf serum at 37 ℃ and 5% CO 2. L02 culture medium plus insulin 0.2μ / ml. HepG_2, SMMC 7721 and L02 cells were infected with HCV positive sera. The three cells were changed every three days, digested and passaged one time. The cells were cultured continuously for 60 days. The nested RT- PCR) to detect positive and negative HCV RNA in cultured cells and supernatant. Results The positive RNA of HCV RNA was detected intermittently in the cells of HepG_2 and SMMC 7721 cells at 2 to 30 days after infection, and the positive RNA of HCV RNA was detected in the cells within 3 to 30 days after infection. The intracellular HCV RNA negative RNA of 3 cells were detected at 3 to 30 days Can be detected intermittently, the detection rate and positive strand RNA close. The positive rate and the negative rate of HCV RNA in HepG2 cells were higher in all the 3 cells. Positive and negative HCV RNA could be detected intermittently on day 31 to day 60, but the degree of replication was weakened. The positive supernatant of HepG_2, SMMC 7721 and L02 cells also showed a positive expression of HCV positive strand RNA. None of the HCV negative strand RNA was detected in the culture supernatant. Conclusion Both HepG_2, SMMC 7721 and L02 cells are susceptible to HCV and can support long-term stable replication of HCV. Therefore, HepG_2 cells are the ideal model of HCV infected cells in vitro.