受控凋亡素基因逆转录病毒载体诱导人肝癌细胞凋亡

Study on Regulated Retroviral Vectors Containing the Apoptin Gene in the Induction of Apoptosis in Human Hepatocellular Carcinoma Cells

目的:构建受控重组凋亡素基因逆转录病毒载体,观察受四环素调控的凋亡素基因的表达对人肝癌细胞的影响。方法:构建重组逆转录病毒表达载体pRevTRE—VP3。调节载体pRevTet-Off和表达载体pRevTRE—VP3分别转染PT67包装细胞后,取其病毒上清液依次感染人肝癌细胞系HepG2,以四环素调控凋亡素基因在HepG2/Off-VP3细胞中的表达,以流式细胞仪检测细胞凋亡率。结果:重组逆转录病毒载体pRevTRE—VP3构建成功;经PCR和RT—PCR证实凋亡素基因在HepG2细胞中得到了表达;HepG2/Off-VP3—8细胞在无四环素培养环境中凋亡率明显高于在1μg/ml四环素环境中的细胞凋亡率(P＜0．05)。结论:凋亡素基因经RevTet系统导入人肝癌细胞HepG2后,可在四环素调控下表达凋亡素并诱导HepG2细胞凋亡。

Objective：To construct a tetracycline-regulated recombinant retroviral vector carrying apoptin gene and investigate the effect of apoptin gene expression on HepG2, a human hepatocellular carcinoma cell line in the control of tetracycline. Methods：The apoptin gene was cloned into a retroviral vector pRevTRE to obtain the recombinant vector pRevTRE-VP3, pRevTRE- VP3 and pRevTet-Off were transfected into PT67 respectively. After antibiotic selection and determining the viral titers, the ceil lines PT67/Off and PT67/VP3 that can stably produce virus were selected. The supernate of PT67/Off and PT67/VP3 containing retroviral vectors was collected and was, in turn, used to infect HepG2 ceils. After antibiotic selection, the resulting cell lines HepG2/Off-VP3 were established. Apoptin gene expression in HepG2/Off-VP3 cells was controlled by the presence or the absence of tetracycline in medium. After AnnexinV-FITC/PI staining, the apoptosis rate in HepG2/Off-VP3 cells was analysed with flow cytometry. Results： pRevTRE-VP3 was identified by restrictive enzyme digestion and sequencing. The inte- gration and expression of apoptin gene in HepG2 was confirmed by PCR and RT-PCR. HepG2/Off-VP3-8 cells were incubated in medium free of tetracycline or in medium with 1 tzg/ml tetracycline for 24 hours, 48 hours and 72 hours. The apoptosis rate in the HepG2/Off-VP3-8 cells incubated in medium free of tetracycline was significant higher than that in the HepG2/Off-VP3- 8 cells incubated in medium with 1μg/ml tetracycline at the same time point. Conclusion：After apoptin, gene is transduced into HepG2 with RevTet system, it can express apoptin and induce apoptosis in HepG2 cells in the control of tetracycline.