腺病毒驱动KDR启动子介导CD/TK融合基因系统对血管内皮细胞的靶向杀伤作用  

Adenovirus mediated fusion gene system driven by KDR promoter kills selectively vein endothelial cells

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

机构地区：[1]第一军医大学珠江医院普通外科,广州510282 [2]第一军医大学肿瘤研究所

出　　处：《中华普通外科杂志》2005年第3期190-193,共4页Chinese Journal of General Surgery

基　　金：国家 8 63计划基金资助项目 (2 0 0 1AA2 17171) ;广东省自然科学基金资助项目 (0 13 0 72 )

摘　　要：目的　探讨腺病毒介导KDR启动子驱动的融合基因体系,对人脐血管内皮细胞ECV30 4的选择性杀伤作用。方法　将质粒pAdEasy -KDR- CDglyTK在2 93细胞内包装、扩增后,体外感染表达KDR的ECV30 4细胞株和不表达KDR的LS174T细胞株,并给予不同浓度的前药5 - 氟胞嘧啶(5 -fuorocytosine ,5 -FC)和/或丙氧鸟苷(ganciclovir,GCV) ,观察该体系对不同细胞株的杀伤效应及其旁观者效应;并以流式细胞仪检测细胞周期的变化,电镜观察细胞的病变。结果　所得病毒对两种细胞细胞的感染率相似,其感染率随腺病毒滴度的递增而增加;表达KDR的ECV30 4细胞对前药的具有较高的敏感性,不表达KDR的LS174T细胞对前药不敏感(P均<0 . 0 1) ;融合基因的疗效优于任一单自杀基因(P均<0 . 0 1) ;且观察到该体系明显的旁观者效应。流式细胞术检测治疗后ECV30 4细胞G1期比率增多及S期细胞减少(P均<0 . 0 1) ,同时,电镜下可见ECV30 4有凋亡和坏死改变。结论　KDR基因启动子可调控融合基因体系选择性杀伤人血管内皮细胞。Objective To evaluate the selectively killing effect of adenovirus (Ad) mediated double suicide gene driven by KDR promoter on vein endothelial ECV304 cells.Methods KDR producing cells ECV304 and the KDR nonproducing cells LS174T were infected by Ad respectively, followed by treatment with 5-FC and GCV.Killing effects were evaluated and bystander effects were analyzed.Distribution of cell cycle was detected by flow cytometric assay and pathological character of cells was observed by electron microscopy.Results The infection rate of the resultant recombinant Ad to all the cells was not apparently different, and it increased gradually with the addition of multiple of infection (MOI) of Ads.ECV304 cells infected with Ad-KDR-CDglyTK were sensitive highly to the prodrugs, but the LS174T cells infected with Ad-KDR-CDglyTK appeared to be unsensitive to the two prodrugs ( P <0.01). The killing effect of CD/TK fusion gene on the target cells was much stronger than that of each suicide gene alone ( P <0.01), and they all had considerable bystander effect.In addition, the cell cycle of ECV304 cells was arrested at G_1 phase and morphologic features of apoptosis and necrosis in ECV304 cells were displayed via electron microscopy.Conclusion The CD/TK fusion gene system controlled by KDR promoter has selectively killing effect on the KDR-CDglyTK expressing endothelial cells.

关 键 词：KDR启动子 靶向杀伤作用 基因系统 驱动 ECV304细胞 LS174T细胞株 选择性杀伤作用 流式细胞仪检测 流式细胞术检测 人血管内皮细胞 旁观者效应 CD 5-氟胞嘧啶 基因体系 腺病毒介导 293细胞 基因启动子 体外感染 不同浓度 丙氧鸟苷 杀伤效应 细胞周期 电镜观察 

分 类 号：R73-36[医药卫生—肿瘤]