KDR介导的双自杀基因重组腺病毒对ECV304细胞增殖活性、细胞周期及凋亡的影响

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (05): 517-520.

KDR介导的双自杀基因重组腺病毒对ECV304细胞增殖活性、细胞周期及凋亡的影响

苏国强¹, 黄宗海¹, 俞金龙¹, 厉周¹, 范应方¹, 宋慧娟¹, 王蔚², 张进华²

1. 南方医科大学珠江医院普外科, 广东, 广州, 510282;
2. 南方医科大学病理教研室, 广东, 广州, 510515

出版日期:2005-05-20 发布日期:2005-05-20
基金资助:
收稿日期:2005-1-12。
基金项目:国家863计划项目(2001AA217171);广东省自然科学基金项目(013072)
作者简介:苏国强(1966- ),男,第一军医大学在读博士研究生,主任医师,电话:020-61643211,E-mail:suguoqiang66@yahoo.con.cn

Effect of KDR recombinant adenovirus containing double suicide gene on proliferation, apoptosis and cell cycle of human umbilical vein endothelial ECV304 cells

SU Guo-qiang¹, HUANG Zong-hai¹, YU Jin-long¹, LI Zhou¹, FAN Ying-fang¹, SONG Hui-Juan¹, WANG Wei², ZHANG Jin-hua²

1. 南方医科大学珠江医院普外科, 广东, 广州, 510282;
2. 南方医科大学病理教研室, 广东, 广州, 510515

Online:2005-05-20 Published:2005-05-20

摘要/Abstract

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株ECV30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ECV30细胞株和对照组不表达KDR的LS17T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ECV30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ECV30细胞及对照组LS17T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ECV30细胞对前药的具有较高的敏感性,与前者相比,不表达KDR的LS17T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ECV30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ECV30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.

Abstract: Objective To study the effect of adenovirus (Ad)-mediated fusion gene systemdriven by KDR promoter on the proliferation, apoptosis and cell cycle of human umbilical vein endothelial ECV304 cells.Methods The KDR-expressing ECV304 cells and LS174T cells not expressing KDR were both infected by the AdEasy-KDR-CDglyTK followed by treatment with the prodrugs 5-flurocytosine (5-FC) and/or ganciclovir (GCV) at different concentrations. The killing effects of the transfection on the cells were evaluated and bystander effects analyzed by coculturing the uninfected cells by AdKDR-CDglyTK with different ratios of infected cells. Flow cytometry was employed for determining the cell cycle distribution and electron microscopy performed to observe the pathological changes of cells.Results The infection rates of the resultant recombinant Ad (rAd) were similar in the cells and gradually increased with the increment in the multiplicity of infection (MOI) of the Ads. The infected cells exhibited different sensitivities to the two prodrugs: ECV304 cells infected with rAd were highly sensitive to the prodrugs, but the infected LS174T cells were not (P<0.001). The killing effect of CD/TK fusion gene on the target cells was much stronger than that of either single suicide gene (P<0.001), showing also obvious bystander effect. In addition, the cell cycle of ECV304 cells was arrested at S phase with morphologic features of apoptosis and necrosis as displayed by electron microscopy.Conclusions CD/TK fusion gene system driven by KDR promoter selectively kills the KDR-CDglyTK-expressing endothelial cells, the mechanism of which may involve cell cycle arrest and necrosis and apoptosis of the cells.

中图分类号:

R730.59

苏国强¹, 黄宗海¹, 俞金龙¹, 厉周¹, 范应方¹, 宋慧娟¹, 王蔚², 张进华². KDR介导的双自杀基因重组腺病毒对ECV304细胞增殖活性、细胞周期及凋亡的影响[J]. 南方医科大学学报, 2005, 25(05): 517-520.

SU Guo-qiang¹, HUANG Zong-hai¹, YU Jin-long¹, LI Zhou¹, FAN Ying-fang¹, SONG Hui-Juan¹, WANG Wei², ZHANG Jin-hua². Effect of KDR recombinant adenovirus containing double suicide gene on proliferation, apoptosis and cell cycle of human umbilical vein endothelial ECV304 cells[J]. Journal of Southern Medical University, 2005, 25(05): 517-520.

参考文献

[1] 谢守珍,王晶,李德忠,等.醋酸甲孕酮对卵巢癌裸鼠抑制瘤的抗血管生成作用[J].第一军医大学学报,2004,24(7):821-3.Xie SZ, Wang J, Li DZ, et al. Medroxyprogesterone acetate therapy against antiangligenesis of transplanted ovarian cancer in nude mice [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2004, 24(7): 821-3.
[2] Ellis LM.A targeted approach for antiangiogenic therapy of metastatic human colon cancer[J]. Am Surg, 2003, 69(1): 3-10.
[3] Yu HK, Ahn JH, Lee HJ, et al. Expression of human apolipoprotein (a) kringles in colon cancer cells suppresses angiogenesis-dependent tumor growth and peritoneal dissemination [J]. J Gene Med, 2005, 7(1): 39-49.
[4] Mullen JT, Donahue JM, Chandrasekhar S, et al. Oncolysis by viral replication and inhibition ofangiogenesis by a replication-conditional herpes simplex virus that expresses mouse endostatin [J]. Cancer,2004, 101(4): 869-77.
[5] Tee D, DiStefano J 3rd. Simulation of tumor-induced angiogenesis and its response to anti-angiogenic drug treatment: mode of drug delivery and clearance rate dependencies [J]. J Cancer Res Clin Oncol, 2004, 130(1): 15-24.
[6] Gora-Kupilas K, Josko J. The neuroprotective function of vascular endothelial growth factor (VEGF)[J]. Folia Neuropathol, 2005, 43 (1): 31-9.
[7] 李斌.体外三维抗血管生成药物筛选模型的建立[J].第一军医大学学报,2004,24(10):1167-70.Li B. Construction of a three-dimensional human angiogenesis model in vitro for antiangiogenic drug selection[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2004, 24(10): 1167-70.
[8] Beierle EA, Dai W, Langham MR Jr, et al. Expression of VEGF receptors in cocultured neuroblastoma cells [J]. J Surg Res. 2004,119(1): 56-65.
[9] Suhardja A, Hoffman H. Role of growth factors and their receptors in proliferation ofmicrovascular endothelial cells [J]. Microsc Res Tech, 2003, 60: 70-5.
[10] VeldwijkMR, FruehaufS, SchiedlmeierB, et al. Differential expression of a recombinant adeno-associated virus 2 vector in human CD34+ cells and breast cancer cells [J]. Cancer Gene Ther,2000,7: 597-604.
[11] 汤福祥,郑权,黄宗海,等.应用改进的AdEasy系统制备重组腺病毒[J].第一军医大学学报,2003,23(5):501-3Tang TX, Zheng Q, Huang ZH, et al. Construction of recombinant adenovirus using modified AdEasy system [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2003, 23(5): 501-3.
[12] 苏国强,黄宗海,刘志锋,等.重组腺病毒驱动KDR-CDglyTK融合基因系统对MCF-7细胞及血管内皮细胞的靶向杀伤作用[J].第一军医大学学报,2004,24(12):1346-9.Su GQ, Huang ZH, Liu ZF, et al. Adenovirus-mediated CDglyTK fusion gene system driven by KDR promoter selectively kills MCF-7 breast cancer cells and vascular endothelial cells [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2004, 24(12): 1346-9.
[13] Kurozumi K, Tamiya T, Ono Y, et al. Apoptosis induction with 5-fluorocytosine/cytosine deaminase gene therapy for human malignant glioma cells mediated by adenovirus [J]. J Neurooncol,2004, 66(1-2): 117-27.