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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株ECV304的增殖活性、细胞周期及凋亡的影响。方法以重组腺病毒AdEasy-KDR-CDglyTK体外感染表达KDR的ECV304细胞株和对照组不表达KDR的LS174T细胞株,并给予不同浓度的前药GCV(ganciclovir)和/或5-FC(5-fluorocytosine),观察该体系对ECV304细胞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。结果重组腺病毒对ECV304细胞及对照组LS174T细胞的感染率相似,其感染率随腺病毒滴度的递增而增加,当MOI为200时,所有细胞株均达约100%感染。以MOI为100的重组体分别感染各细胞株表现出对前药的不同敏感性:表达KDR的ECV304细胞对前药的具有较高的敏感性,与前者相比,不表达KDR的LS174T细胞对前药不敏感(P均<0.001)。融合基因的疗效优于任一单自杀基因(P均<0.001)。将感染腺病毒的细胞与未感染细胞以不同混合培养,观察到该体系明显的旁观者效应。流式细胞术检测表明该体系抑制ECV304细胞DNA的合成,表现为S期细胞比率增多及G1期细胞减少(P均<0.001)。同时,电镜下可见ECV304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,其机制与细胞周期阻滞、凋亡及坏死有关。
Objective To investigate the effect of adenovirus-mediated KDR promoter-driven fusion gene on the proliferation, cell cycle and apoptosis of human umbilical cord blood endothelial cell line ECV304. Methods In vitro infection of KDR-expressing ECV304 cells with recombinant adenovirus AdEasy-KDR-CDglyTK and LS174T cells without KDR in the control group were treated with different concentrations of ganciclovir and / or 5-fluorocytosine ). The killing effect of ECV304 cells and its bystander effect were observed. The changes of cell cycle were detected by flow cytometry and the cell lesions were observed by electron microscope. Results The infection rate of recombinant adenovirus was similar to that of LS174T cells in ECV304 cells and control group. The infection rate increased with the increase of adenovirus titer. When the MOI was 200, all cell lines reached about 100% infection. Infection of each cell line with a recombinant having an MOI of 100 showed different sensitivities to prodrugs: ECV304 cells expressing KDR were highly sensitive to prodrugs compared to the LS174T cells that did not express KDR Prodrugs are not sensitive (P <0.001). The efficacy of the fusion gene is superior to that of any single suicide gene (P <0.001). The adenovirus-infected cells were cultured in different mixes with uninfected cells, and the obvious bystander effect of this system was observed. Flow cytometry showed that the system inhibited the DNA synthesis of ECV304 cells. The results showed that the proportion of cells in S phase increased and the number of cells in G1 phase decreased (P <0.001). Meanwhile, ECV304 showed apoptosis and necrosis changes under electron microscope. Conclusion KDR gene promoter can regulate the fusion gene system selectively kill human vascular endothelial cells, the mechanism of which is related to cell cycle arrest, apoptosis and necrosis.