腺病毒介导双自杀基因选择性杀伤血管内皮细胞  

Adenovirus mediated double suicide gene selectively kills human umbilical vein endothelial cells

作　　者：杨文宇[1] 黄宗海[1] 龚小卫[2] 程琦[3] 车小燕[4] 

机构地区：[1]第一军医大学珠江医院普通外科,广州510282 [2]第一军医大学病理生理教研室 [3]广州军区总医院超声诊断室 [4]第一军医大学珠江医院中心实验室,广州510282

出　　处：《中华胃肠外科杂志》2004年第1期65-68,共4页Chinese Journal of Gastrointestinal Surgery

基　　金：广东省自然科学基金重点资助项目(013072);国家863计划项目(2001AA217171)

摘　　要：目的研究腺病毒介导的双自杀基因在KDR启动子调控下对血管内皮细胞的选择性杀伤作用。方法应用PCR扩增出KDR启动子序列,分别构建携带KDR启动子和巨细胞病毒(CMV)启动子调控下的CD与TK的融合基因的重组腺病毒AdKDR-CDglyTK、AdCMV-CDglyTK,体外感染脐静脉血管内皮细胞系HUVEC和结直肠癌细胞系LOVO细胞,利用重组病毒携带的GFP基因,在荧光显微镜下观测重组病毒的感染效率,并给予不同浓度的GCV和5-氟胞嘧啶(5-FC),观测杀伤作用,比较两种启动子的转录调控特性。结果成功构建了两种重组病毒,并高效地转染了HUVEC和LOVO细胞。两种重组病毒对两种细胞的转染效率相似,并随重组病毒的感染复数MOI(multiplicityofinfection)增加而升高;以MOI=100的两种病毒分别转染的两种细胞表现出对前药的不同敏感特性:转染了AdCMV-CDglyTK的HUVEC和LOVO细胞以及转染了AdKDR-CDglyTK的HUVEC细胞,对前药的敏感性差异无显著性意义(P>0.05);转染了AdKDR-CDglyTK的LOVO细胞,对前药敏感性低,与其他3组比较,差异存在显著性意义(与其他3组两两间比较,P均<0.001)。结论KDR基因启动子可调控双自杀基因在血管内皮细胞中的特异性表达,有利于实现双自杀基因靶向恶性肿瘤血管内皮细胞的抑癌疗法。Objective To study the selectively killing effect of adenovirus mediated double suicide gene under the regulation of KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter was cloned by PCR, and two recombinant adenoviral plasmid pAdKDR CDglyTK,pAdCMV CDglyTK were constructed in a “two step transformation protocol”. These two newly constructed plasmids were then transfected to 293 packaging cells to grow adenovirus. Human umbilical vein endothelial cells (HUVEC) and LOVO cells were infected with either of the two resultant recombinant adenovirus(AdKDR CDglyTK and AdCMV CDglyTK) respectively. The infection rate was measured with the aid of GFP expression. Infected cells were cultured in mediums containing different concentration of GCV and 5 FC, and the killing effects were measured. Results Two recombinant adenoviral plasmids pAdKDR CDglyTK, pAdCMV CDglyTK were successfully constructed and transfected into 293 cells. The infection rates of the two resultant recombinant adenovirus to the two cell lines were similar. The infected cells exhibited different sensibility to the prodrugs:HUVEC cells and LOVO cells infected with AdCMV CDglyTK, and HUVEC cells infected with AdKDR CDglyTK were highly sensitive to the prodrugs. There were no significant differences among them. The sensitivity of LOVO cells infected with AdKDR CdglyTK to the prodrugs was lower than those of the former cells(P< 0.001). Conclusion Selective killing of HUNEC cells may be achieved by gene transfer of double suicide gene under regulation of KDR promoter, which may provide an optional way to target gene therapy of malignancy.

关 键 词：腺病毒 自杀基因 选择性杀伤 血管内皮细胞 KDR启动子 

分 类 号：R73-36[医药卫生—肿瘤]