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目的建立SR-BI表达抑制的肝癌细胞系,以作为研究SR-BI基因突变对HCV感染性影响的细胞模型。方法构建SR-BI短发夹状RNA(Short hairpin RNA,sh RNA)慢病毒重组质粒Lv-SR-BI-sh RNA,挑取克隆进行PCR扩增和序列测定;将阳性重组质粒与慢病毒辅助质粒共转染HEK2973T细胞,包装SR-BI sh RNA慢病毒并进行病毒滴度测定。SR-BI sh RNA慢病毒感染人肝癌细胞株Huh7和Huh7.5.1细胞,嘌呤霉素筛选,Real-time PCR和Western blot法检测SR-BI m RNA转录和蛋白表达。结果测序结果证实Lv-SR-BI-sh RNA慢病毒载体构建成功并获得HEK293T细胞中包装的慢病毒,Lv-SR-BI-sh RNA重组慢病毒感染Huh7和Huh7.5.1细胞,感染组SR-BI m RNA转录降低,蛋白表达降低。结论建立了SR-BI表达抑制的人肝癌细胞株Huh7-si SR-BI和Huh7.5.1-si SR-BI稳定细胞系。
Objective To establish a hepatocellular carcinoma cell line with suppressed expression of SR-BI as a cell model to study the effect of SR-BI mutation on HCV infectivity. Methods The SR-BI short hairpin RNA (sh RNA) lentivirus recombinant plasmid Lv-SR-BI-sh RNA was constructed and cloned for PCR amplification and sequencing. The positive recombinant plasmids were supplemented with lentivirus Plasmids were co-transfected into HEK2973T cells and packaged with SR-BI sh RNA lentivirus for virus titers. SR-BI sh RNA lentivirus was used to infect human hepatoma Huh7 and Huh7.5.1 cells. The puromycin was screened by puromycin, and the transcription and protein expression of SR-BI m RNA were detected by Real-time PCR and Western blot. Results The sequencing results confirmed that the lentiviral vector of Lv-SR-BI-sh RNA was successfully constructed and got the lentivirus packaged in HEK293T cells. The Lv-SR-BI-sh RNA recombinant lentivirus infected Huh7 and Huh7.5.1 cells, BI m RNA transcription decreased, protein expression decreased. Conclusion The human hepatoma cell line Huh7-si SR-BI and Huh7.5.1-si SR-BI stable cell lines with SR-BI expression inhibition were established.