SP-TAT-Apoptin融合蛋白对HepG2细胞周期的影响  

SP-TAT-Apoptin induces G1 arrest in HepG2 cells

作　　者：韩苏夏[1] 马瑾璐[1] 吕毅[1] 黄辰[2] 段康民[3] 

机构地区：[1]西安交通大学医学院第一附属医院肿瘤中心,陕西西安710061 [2]西安交通大学医学院中心实验室,陕西西安710061 [3]西北大学生命科学院分子微生物实验室,陕西西安710069

出　　处：《细胞与分子免疫学杂志》2008年第9期864-866,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30672069)

摘　　要：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 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- VS-D-TOPO. 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 supematant was collected. Then the expression of the fusion protein was detected by RT- PCR and Western blot. HepG2 cells were co-cultured with the supematant. 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.

关 键 词：信号肽 蛋白转导域 鸡贫血病毒 Apoptin蛋白 肝癌 

分 类 号：R735[医药卫生—肿瘤]