AdKDR-CDglyTK系统对结直肠癌细胞及血管内皮细胞的靶向杀伤作用  

Adenovirus mediated double suicide gene under controlling by KDR promoter selectively kills vascular endothelial cells and colorectal tumor cells

作　　者：黄宗海[1] 苏国强[1] 杨祖立[2] 俞金龙[1] 厉周[1] 范应方[1] 宋慧娟[1] 

机构地区：[1]南方医科大学珠江医院普通外科,广州510282 [2]中山大学附属第一医院胃肠胰外科

出　　处：《中华胃肠外科杂志》2005年第2期147-150,共4页Chinese Journal of Gastrointestinal Surgery

基　　金：国家863计划项目(2001AA217171);广东省自然科学基金项目(013072)

摘　　要：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 investigate the selectively killing effect of adenovirus (Ad) mediated double suicide gene under the regulation of KDR promoter on vascular endothelial cells and colorectal tumor cells. Methods 293 packaging cells were transfected with the plasmids of pAdEasy KDR CDglyTK and pAdEasy CMV CDglyTK and the infectious viruses were generated. The KDR expressive cells of ECV304,SW620 and the KDR inexpressive cells of LS174T were infected by two Ads. The infection rate was observed and the expression of CDglyTK was detected by RT PCR. After treatment with different concentrations of 5 FC and GCV,the killing effect and bystander effect on ECV304,SW620 and LS174T were examined. Results The titers of these two purified Ads were 2.0×1012 pfu/ml. There was no significant difference in infection rate between two recombinant Ads infecting various cells,and the infection rate increased in accordance with the enhancing titers of Ads. RT PCR demonstrated that there existed the product of CDglyTK gene in all the cells infected by Ad CMV CDglyTK and the cells infected by Ad KDR CDglyTK except in the SL174T. The curative effect in this system on various cells was shown as follows:(1)All cells infected with Ad CMV CDglyTK and some cells of ECV304 and SW620 infected with Ad KDR CDglyTK were highly sensitive to the prodrugs,but there was no significant differences among them(P >0.05); compared with ECV304 and SW620 cells,LS174T cells were not sensitive to the two prodrugs(P< 0.001). (2) The efficacy of double suicide gene was better than that of single suicide gene(P< 0.001). (3)The system had considerable bystander effect. Conclusion The double suicide gene under the regulation of KDR promoter has specific killing effect on the KDR expressing endothelial cells and colorectal tumor cells.

关 键 词：KDR 感染 结直肠癌 表达 细胞株 双自杀基因 T细胞 启动子 目的基因 重组体 

分 类 号：R735.3[医药卫生—肿瘤]