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目的研究腺病毒介导的血管内皮细胞生长因子受体(KDR)启动子驱动CD/TK双自杀基因系统对血管内皮细胞及结直肠癌肿瘤细胞的选择性杀伤作用。方法质粒pAdEasy-KDR-CDglyTK和pAdEasy-CMV-CDglyTK在293细胞中包装、扩增后,体外感染表达KDR的ECV304、SW620细胞株和不表达KDR的LS174T细胞株,观察其感染效率并以RT-PCR方法检测转基因细胞CDglyTK的表达,然后给予不同浓度的前药5-氟胞嘧啶(5-FC)及更昔洛韦(GCV)处理,观察该体系对不同细胞株的杀伤效应及其旁观者效应。结果两种病毒滴度均为2.0×1012pfu/ml。两种重组体对各细胞株的感染率相似,其感染率随腺病毒滴度的增高而递增。RT-PCR方法检测发现:除感染AdKDR-CDglyTK的LS174T细胞外,感染AdCMV-CDglyTK的所有细胞及感染AdKDR-CDglyTK的其他两种细胞均有目的基因CDglyTK的表达。该体系治疗结果提示:(1)感染AdCMV-CDglyTK的所有细胞株和感染AdKDR-CDglyTK的ECV304、SW620细胞对前药具有较高的敏感性,且其敏感性差异无显著性意义(均P>0.05),与前两者相比感染AdKDR-CDglyTK的LS174T细胞对前药不敏感(均P<0.001);(2)双自杀基因的疗效优于任一单自杀基因的疗效(均P<0.001);(3)该体系旁观者效应明显。结论KDR基因启动子可调控双自杀基因系统选择性杀伤表达KDR的血管?
Objective To study the selective killing effect of adenovirus-mediated vascular endothelial growth factor receptor (KDR) promoter-driven CD / TK suicide gene system on vascular endothelial cells and colorectal cancer cells. Methods The plasmids pAdEasy-KDR-CDglyTK and pAdEasy-CMV-CDglyTK were packaged in 293 cells and amplified. The KDR-expressing ECV304 and SW620 cells and LS174T cells without KDR were infected in vitro. The infection efficiency was observed by RT- PCR method was used to detect the expression of CDglyTK in transgenic cells, then treated with different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV) to observe the killing effect of this system on different cell lines and their bystanders effect. Results Both virus titers were 2.0 × 1012 pfu / ml. The infection rate of the two recombinants to each cell line was similar, and the infection rate increased with the increase of adenovirus titer. The results of RT-PCR showed that all the cells infected with AdCMV-CDglyTK and the other two cells infected with AdKDR-CDglyTK had the expression of CDglyTK, except LS174T cells infected with AdKDR-CDglyTK. The results showed that: (1) All cell lines infected with AdCMV-CDglyTK and ECV304 and SW620 cells infected with AdKDR-CDglyTK had higher sensitivity to prodrugs and no significant difference in sensitivity (P> 0.05). LS174T cells infected with AdKDR-CDglyTK were not sensitive to prodrugs compared with the former two groups (all P <0.001). (2) The efficacy of double suicide gene was superior to that of any single suicide gene ); (3) the system bystander effect is obvious. Conclusion KDR gene promoter can regulate double suicide gene system to selectively kill the blood vessels that express KDR?