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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。
Objective To study the killing effect of adenovirus-mediated dual suicide gene system of KDR / CD / TK on SW480 cells. Methods The recombinant adenovirus pAdKDR / CD / TK was constructed and transfected into SW480 cells, ECV304 cells and LS174T cells with recombinant adenovirus of different multiplicity of infection (MOI), and then cultured in different concentrations of prodrugs (GCV, 5-FC) The cytotoxicity of the recombinant adenovirus combined with GCV and 5-FC on the cells of colorectal cancer SW480 cells, ECV304 cells and LS174T cells was observed by MTT assay, and the effect of GCV, 5-FC and GCV / 5- Killing effect of the kind of cells and targeted killing effect of KDR promoter. Results The recombinant adenovirus of pAdKDR / CD / TK had similar infection rates to all three kinds of cells. The expression of the target gene was detected in all three kinds of cells infected with adenovirus except LS174T cells. When the ratio of transgenic cells was 50%, the survival rates of SW480 and ECV304 cells were (31.3 ± 5.64)% and (33.5 ± 5.4)%, respectively, while those of LS174T cells were still (98.1 ± 0.95)% ). 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)% at the GCV concentration of 80μg / ml, The survival rates were (40.4 ± 5.8)%, (45.3 ± 4.7)%, (97.0 ± 6.2)% and 80μg / ml + 1000μg / ml respectively (20.5 ± 0.46)%, (25.6 ± 1.33)% and (97.3 ± 3.96)%, respectively. The combined treatment with GCV and 5-FC alone, respectively, the survival rate, the differences were statistically significant (P all <0.05). Prompt application of GCV alone and 5-FC on SW480 cells, ECV304 cells have a stronger killing effect, the combined effect is better. Conclusion The fusion gene containing KDR promoter has a selective killing effect. The prodrug GCV and 5-FC could inhibit the proliferation of colorectal cancer SW480 cells transfected with fusion gene in vitro and had a strong bystander effect.