pAdKDR/CD/TK自杀基因系统对大肠癌SW480细胞体外杀伤作用的实验研究

目的研究腺病毒介导的KDR/CD/TK双自杀基因系统对大肠癌SW480细胞的杀伤作用。方法构建pAdKDR/CD/TK重组腺病毒,采用不同感染复数（MOI）的重组腺病毒感染大肠癌SW480细胞、ECV304细胞及LS174T细胞,再加入不同浓度前药（GCV、5-FC）培养后,以MTT法检测细胞生长抑制率,观察重组腺病毒联合GCV和5-FC对大肠癌SW480细胞、ECV304细胞和LS174T细胞的杀伤作用,了解前药GCV、5-FC、GCV/5-FC对3种细胞的杀伤作用及KDR启动子的靶向杀伤作用。结果 pAdKDR/CD/TK重组腺病毒对3种细胞具有相似的感染率,感染腺病毒的3种细胞中除LS174T细胞外,均检测到目的基因的表达。当转基因细胞的比率为50%时,SW480和ECV304细胞分别有（31.3±5.64）%、（33.5±5.4）%的存活,而LS174T细胞存活率仍为（98.1±0.95）%（P均〈0.01）。单用GCV浓度为80μg/ml时,SW480细胞、ECV304细胞、LS174T细胞的生存率分别为（45.6±6.5）%、（45.9±5.4）%、（99.5±4.7）%,单用5-FC浓度为1000μg/ml时,三者生存率分别为（40.4±5.8）%、（45.3±4.7）%、（97.0±6.2）%,联合用药（80μg/ml＋1000μg/ml）时,三者生存率分别为（20.5±0.46）%、（25.6±1.33）%、（97.3±3.96）%。联合用药分别与GCV及5-FC单独用药生存率比较,差异均有统计学意义（P均〈0.05）。提示单独应用GCV及5-FC对SW480细胞、ECV304细胞有较强的杀伤作用,联合用药效果更佳。结论含KDR启动子的融合基因,具有选择性杀伤作用。前药GCV、5-FC对转染融合基因的大肠癌SW480细胞具有体外抑制作用,且有较强的旁观者效应。

Killing effect of KDR promoter driving double suicide gene on human colorectal cancer SW480 cells in vitro

Objective To investigate the killing effect of adenovirus vector containing KDR promoter driving double suicide genes on colorectal SW480 cells in vitro.Methods The pAdKDR/TK/CD re-compound adenovirus vector was constructed and the SW480 colorectal cancer cells,ECV304 cells and LS174T cells were transferred by pAdKDR/TK/CD re-compound adenovirus of different multiplicity of infection（MOI）.Then the pro-drugs （GCV,5-FC） of different concentration were added in them for co-culturing.The cell growth inhibiting rate was detected by MTT method for observing killing effect of pro-drug and understanding the target killing effect of KDR promoter.Results The pAdKDR/TK/CD re-compound adenovirus had similar transferring efficiency to SW480,ECV304 and LS174T cells.The expres sions of target gene in transferred cells were all detected besides LS174T cells.When the ratio of transferred cells was 50%,the survival rates were （31.3±5.64）% for SW480 cells,（33.5±35.4）% for ECV304 cells and （98.1±0.95）% for LS174T cells.The former two did not have significant difference （P〉0.05）,but they were all significantly different from the latter （all P〈0.01）.When the concentration of the GCV was 80 μg/ml,the survival rates of SW480 cells,ECV304 cells and LS174T cells were （45.6±6.5）%,（45.9±5.4）% and （99.5±4.7）%,respectively;when the concentration of 5-FC was 1 000 μg/ml,the survival rates of three kind cells were （40.4±5.8）%,（45.3±4.7）% and （97.0±6.2）%,respectively;when combining GCV with 5-FC（80/1 000 μg/ml）,the survival rates of three kind cells were（20.5±0.5）%,（25.6±1.3）% and （97.3±4.0）%,respectively.Compared with GCV or 5-FC alone,the survival rates of SW480 and ECV304 cells were obviously lower（all P〈0.05）in their combining application.Conclusions The fusion gene built up with KDR promoter has selective killing effect.The prodrugs GCV and 5-FC have killing effect on SW480 cells transfected by fusion gene in vitro,and have stronger bystander effect.