纳米磁流体介导的重组质粒PEGFP-AFP-hTNFα对肝癌细胞HepG2的体外杀伤作用  

Recombinant plasmid PEGFP-AFP-hTNFαdelivered by nano-magnetic fluids killed liver cancer cells (HepG2 cells) in vitro

作　　者：彭健[1] 曾杰[1] 刘美洲[1] 邹芬[1] 刘路[1] 汤伟[1] 周健[1] 

机构地区：[1]湘雅医院卫生部纳米生物技术重点实验室,湖南长沙410008

出　　处：《中国现代医学杂志》2008年第7期847-851,共5页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30672047);中国博士后科学基金(No:20060390884);湖南省博士后科学基金(No:2006FJ4240)

摘　　要：目的探讨纳米磁流体作为肝癌基因治疗载体的可行性及AFP增强子能否使外来基因在AFP阳性细胞内高表达。方法构建重组质粒PEGFP-AFP-hTNFα并通过纳米磁流体分别转染入AFP表达阳性的肝癌细胞HepG2及AFP表达阴性的宫颈癌细胞Hela。转染12h后,于荧光显微镜下动态观察;48h后,RT-PCR和Westernblot分析HepG2细胞中hTNFα基因的表达情况,用MTT法检测HepG2细胞的存活,用流式细胞分析HepG2细胞的凋亡。结果纳米磁流体能将重组质粒PEGFP-AFP-hTNFα转入HepG2细胞,并且其转染效率高于脂质体;RT-PCR及Westernblot证明转染的HepG2细胞有hTNFα基因的表达;MTT及流式细胞分析说明hTNFα基因发挥杀伤细胞作用。结论纳米磁流体能将重组质粒PEGFP-AFP-hTNFα转染HepG2细胞并获得表达,可作为肝癌基因治疗的基因载体。采用AFP增强子可使目的基因在HepG2细胞中定向而特异性表达。[Objective] To identify the possibility of using nano-magnetic fluids as gene vector for liver cancer gene therapy. To study the effect of AFP enhancer on tumor-specific expression of foreign gene in AFP-producing cancer cells like HepG2 cells. [Methods] We constructed onstruct recombinant plasmid PEGFP-AFP-hTNFα which was delivered by nano-magnetic fuilds into AFP positive HepG2 cells and AFP negative Hela cells. The fluorescence was detected in order to evaluate the transfection rate after transfecting 12 hs. RT-PCR and Western blot were used to detect expression of hTNFα gene at 48 h after transfection. MTT assay was used to evaluate the effect of hTNFαon the proliferation of HepG2 cells, Flow cytometry was used to analyze the apoptose of HepG2 cells, [Results] Nano-Magnetic fluids delivered plasmid PEGFP-AFP-hTNFα into HepG2 cells and hTNFα gene was successfully expressed. The transfection efficacy of nano-magnetic fluids was superior than lipofectamine in HepG2 cells. RT-PCR and Western blot demonstrated that hTNFαgene was expressed in HepG2 cells after being transfected with nano-magnetic fluids/PEGFP-AFP-hTNFα. MrIT and flow cytometry showed hTNFα gene exerts cellkilling effect, [Conclusions] Nano-Magnetic fluids could successfully deliver PEGFP-AFP-hTNFα into HepG2 cells and induce expression of hTNFα, it is promising gene vector for liver cancer gene therapy. AFP enhancer can specifically enhance the expression of target gene within the cell with positive AFP.

关 键 词：纳米磁流体 AFP增强子 hTNFα基因 肝癌 基因治疗 

分 类 号：R318[医药卫生—生物医学工程]