SP-TAT-Apoptin融合蛋白对HepG2细胞周期的影响

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.

SP-TAT-Apoptin induces G1 arrest in HepG2 cells

AIM: To investigate the effect of SP-TAT-Apoptin in inducing HepG2 cells apoptosis and the possible application on hepatocellular carcinoma gene therapy. METHODS: Recombinant gene SP-TAT-Apoptin was amplified by PCR and cloned into the eukaryotic vector plenti6-V5-D-TOPO(R). After the recombinant plasmid was identified by restriction enzyme digesttion analysis and DNA sequencing, CHO cells were stably transfected with SP-TAT-Apoptin gene and the culture supernatant was collected. Then the expression of the fusion protein was detected by RT-PCR and Western blot. HepG2 cells were co-cultured with the supernatant. At various times post co-culture, HepG2 cells were detected by FCM. RESULTS: The secretory Tat-Apoptin has an additive by-stander effect as an anti-cancer therapy in vitro. The recombinant Apoptin was able to be secreted from transfected cells and re-enter adjacent untransfected HepG2 cells, it can induce HepG2 cells apoptosis and induce G0/G1 arrest. CONCLUSION: SP-TAT-Apoptin can induce HepG2 cell apoptosis and cell cycle G1 arrest.