Double suicide genes selectively kill human umbilical vein endothelial cells.
Double suicide genes selectively kill human umbilical vein endothelial cells.
复制标题
双自杀基因选择性杀死人脐静脉内皮细胞
DOI：
10.1186/1743-422x-8-74
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发表时间：
2011-02-21
期刊：
影响因子：
4.8
通讯作者：
Che G
中科院分区：
文献类型：
--
作者：
Jia W;Mei L;Wang Y;Liu L;Che G
目的构建含CDlyTK双自杀基因的重组腺病毒载体，评价KDR启动子驱动的双自杀基因对人脐静脉内皮细胞的杀伤作用。方法采用聚合酶链式反应(PCR)技术克隆人KDR启动子、大肠杆菌胞嘧啶脱氨酶(CD)基因和单纯疱疹病毒胸苷激酶(TK)基因。构建含KDR启动子和CDlyTK基因的重组表达载体pKDR-CDlyTK。构建了重组腺病毒载体AdKDR-CDlyTK，并将其导入293包装细胞，培养并收获腺病毒。以不同的感染复数(MOI)感染表达KDR的人脐静脉内皮细胞(ECV304)和KDR阴性的肝癌细胞株(HepG2)。通过绿色荧光蛋白(GFP)的表达来衡量感染率。感染细胞在含有不同浓度更昔洛韦(GCV)和/或5-氟胞嘧啶(5-FC)的培养液中培养。用Annexin V-FITC染色和末端转移酶介导的dUTP缺口末端标记(TUNEL)两种不同的方法检测其杀伤效果。结果成功构建重组腺病毒AdKDR-CDlyTK，并有效地感染ECV304和HepG2细胞。感染率与重组腺病毒的MOI有关。感染AdKDR-CDlyTK的ECV304细胞对GCV和5-FC高度敏感。细胞存活率依赖于前药浓度和重组腺病毒的MOI。反之，对HepG2细胞无杀伤作用。两种前药联合应用对ECV304细胞的杀伤作用明显优于单用GCV或5-FC。结论AdKDR-CDlyTK/Double Prodrg系统可能是一种抑制肿瘤血管生成的有效方法。
BackgroundTo construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells.MethodsHuman KDR promoter,Escherichia coli(E. coli) cytosine deaminase (CD) gene and the herpes simplex virus-thymidine kinase (TK) gene were cloned using polymerase chain reaction (PCR). Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304) and KDR-negative liver cancer cell line (HepG2) were infected with the recombinant adenoviruses at different multiplicity of infection (MOI). The infection rate was measured by green fluorescent protein (GFP) expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV) and/or 5-fluorocytosine (5-FC). The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL) staining.ResultsRecombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs.ConclusionAdKDR-CDglyTK/double prodrog system may be a useful method for suppressing tumor angiogenesis.