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目的:研究SP-TAT-Apoptin融合基因诱导人肝癌HepG2细胞凋亡和细胞周期阻滞,探讨其用于肝癌治疗的可能性。方法:通过DNA重组技术,以pcDNA3.1/Apoptin质粒11为模板,构建SP-TAT-Apoptin融合基因plenti6-V5-D-TOPO真核表达载体。用SP-TAT-Apoptinplenti6-V5-D-TOPO真核表达载体转染中国仓鼠卵巢细胞(CHO),转染后Blasticidin霉素进行筛选,并进行鉴定得到稳定表达SP-TAT-Apoptin的CHO细胞株。收集含有TAT-Apoptin融合蛋白的筛选细胞培养上清,用于HepG2细胞培养。于共培养后的不同时段收集HepG2细胞,流式细胞术(FCM)检测细胞凋亡和细胞周期。结果:用包含SP-TAT-Apoptin融合基因的plenti6-V5-D-TOPO真核表达载体瞬时转染中国仓鼠卵巢细胞(CHO),成功筛选出稳定表达SP-TAT-Apoptin融合蛋白的CHO细胞,收集细胞培养上清,继续用于HepG2细胞培养,可观察到HepG2细胞阻滞于G1期并引起细胞凋亡。结论:SP-TAT-Apoptin融合表达可引起HepG2细胞的细胞周期G1期阻滞。
AIM: To investigate the apoptosis and cell cycle arrest induced by SP-TAT-Apoptin fusion gene in HepG2 human hepatocellular carcinoma (HCC) and to explore the possibility of its application in the treatment of liver cancer. Methods: The eukaryotic expression vector plenti6-V5-D-TOPO with SP-TAT-Apoptin fusion gene was constructed by pcDNA3.1 / Apoptin plasmid 11 as template. Chinese hamster ovary cells (CHO) were transfected with the SP-TAT-Apoptinplenti6-V5-D-TOPO eukaryotic expression vector. After transfection, Blasticidin was screened and identified. CHO cell lines stably expressing SP-TAT-Apoptin . Screening cell culture supernatants containing TAT-Apoptin fusion protein were collected for HepG2 cell culture. HepG2 cells were collected at different time points after co-culture, and apoptosis and cell cycle were detected by flow cytometry (FCM). Results: Chinese hamster ovary cells (CHO) were transiently transfected with the plenti6-V5-D-TOPO eukaryotic expression vector containing SP-TAT-Apoptin fusion gene. CHO cells stably expressing the fusion protein SP-TAT- The cell culture supernatants were collected and continued to be used for HepG2 cell culture. It was observed that HepG2 cells arrested in the G1 phase and caused apoptosis. Conclusion: The fusion of SP-TAT-Apoptin can cause cell cycle G1 arrest in HepG2 cells.