FCU1重组腺病毒载体构建及对结肠癌细胞的杀伤作用  

Construction of FCU1 recombinant adenovirus vector and its killing effect on colon cancer cells

作　　者：吴斌文[1] 李友佳[1] 张凯军[1] 李东风[1] 曾志刚[1] 张瑛华[1] 耿庆山[1] 

机构地区：[1]广东省人民医院东病区消化内科广东省老年医学研究所,广东省广州市510080

出　　处：《世界华人消化杂志》2008年第23期2599-2603,共5页World Chinese Journal of Digestology

摘　　要：目的:构建含融合自杀基因FCU1的重组腺病毒载体,转染293细胞构建重组腺病毒,并感染结肠癌细胞,同时给以前体药物5-FC,观察其对结肠癌细胞的杀伤作用.方法:将FCU1基因采用EcoRⅠ、SalⅠ双酶切克隆至穿梭质粒pDC316,构建质粒pDC316-FCU1,并酶切和测序鉴定.pDC316-FCU1与骨架质粒pBHG经脂质体共转染HEK293细胞,在HEK293细胞中同源重组并包装重组腺病毒Ad5-FCU1.将Ad5-FCU1感染结肠癌细胞HCT116,同时给以前体药物5-FC,MTT法检测5-FC对HCT116杀伤作用.结果:重组质粒pDC316-FCU1构建成功.pDC316-FCU1与骨架质粒pBHG共转染HEK293细胞后,出现明显的毒斑,纯化后病毒滴度为2×1012PFU/L.重组病毒Ad5-FCU1感染HCT116细胞后,给以无毒前体药物5-FC显示极强的杀伤作用,当5-FC达100μmol/L时,存活的HCT116/FCU1细胞极少,而对照组存活率接近90%.结论:成功构建了含融合自杀基因FCU1的Ad5-FCU1重组腺病毒,FCU1/5-FC自杀基因系统对结肠癌细胞具有显著的体外杀伤作用.AIM： To construct a recombinant adenovirus vector containing fusion suicide gene FCU1, and study the cytotoxic effect of prodrug 5-FC on colon cancer cells. METHODS： Restriction fragment of FCU1 was inserted into pDC316 vector with EcoR I and Sal I. Recombinant plasmid pDC316-FCU1 was confirmed by restriction enzyme digestion and sequencing. Thereafter, pDC316-FCU1 was co- transfected with adenovirus backbone pBHG into 293 cells to establish the recombinant adenovirus Ad5-FCU1 by homologous recombination. The cytotoxic effect of prodrug 5-FC on the infected HCT116 cells was determined with MTF assay.RESULTS： Recombinant plasmid pDC316- FCUlwas constructed successfully. After pDC316-FCU1 together with adenovirus backbone pBHG was cotransfected into 293 cells, significant virus plaques were observed, which showed the successful homologous recombination and virus packaging in 293 cells. The titer of the purified Ad-FCU1 was 2 × 10^12 PFU/L. Nontoxic prodrug 5-FC has significant cytotoxic effect on HCT116 cells infected with pAd5- FCU1. The survival of HCT116/FCU1 cells was minimal, while that of the control cells was close to 90%, when 5-FC was used at 100 μmol/L. CONCLUSION： Recombinant adenovirus Ad5- FCU1 containing fusion suicide gene FCU1 is established successfully, which has significant killing effect on colon cancer cells in vitro.

关 键 词：结肠癌 融合自杀基因 基因治疗 

分 类 号：R735.35[医药卫生—肿瘤]