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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 investigate the selective killing of human umbilical vein endothelial cells (ECV30 4) by adenovirus mediated KDR promoter-driven fusion gene system. Methods Plasmid pAdEasy-KDR-CDglyTK was packaged in 293 cells and amplified. In vitro, KDR-expressing ECV304 cells and KDR-deficient LS174T cells were infected with various concentrations of prodrug 5-fluorocytosine 5-fluorouracil, 5-fluorouracil and 5-fluorouracil) and / or ganciclovir (GCV). The cytotoxicity and bystander effect of this system on different cell lines were observed. The changes of cell cycle were detected by flow cytometry. Observation of cell lesions. Results The infection rates of the two kinds of cells were similar, and the infection rate increased with the increase of adenovirus titer. ECV30 4 cells expressing KDR were highly sensitive to prodrugs, and LS174T cells that did not express KDR The prodrugs were insensitive (P <0.01). The efficacy of the fusion gene was superior to that of any single suicide gene (P <0.01). A significant bystander effect was observed in this system. Flow cytometry showed that the ratio of G1 phase of ECV304 cells and the number of S phase cells were decreased after treatment (all P <0.01). Meanwhile, apoptosis and necrosis of ECV30 4 cells were observed under electron microscope. Conclusion The promoter of KDR gene can regulate the fusion gene system to selectively kill human vascular endothelial cells.