Department of Thoracic Surgery, West China Hospital, Sichuan University, Chengdu 610041, PR China.
Virol J. 2011 Feb 21;8:74. doi: 10.1186/1743-422X-8-74.
To 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.
Human 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.
Recombinant 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.
AdKDR-CDglyTK/double prodrog system may be a useful method for suppressing tumor angiogenesis.
构建含有 CDglyTK 双自杀基因的重组腺病毒，评价激酶结构域插入受体（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。
应用聚合酶链反应（PCR）技术克隆人 KDR 启动子、大肠埃希菌胞嘧啶脱氨酶（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 CDglyTK 基因的质粒 pKDR-CDglyTK，构建重组腺病毒质粒 AdKDR-CDglyTK，转染 293 包装细胞生长收获腺病毒。用不同感染复数（MOI）感染表达 KDR 的人脐静脉内皮细胞（ECV304）和不表达 KDR 的肝癌细胞系（HepG2），通过绿色荧光蛋白（GFP）表达检测感染率。用含不同浓度前体药物更昔洛韦（GCV）和/或 5-氟胞嘧啶（5-FC）的培养基培养感染细胞，用两种不同方法即 Annexin V-FITC 染色和末端转移酶介导的 dUTP 缺口末端标记（TUNEL）染色检测杀伤作用。
成功构建重组腺病毒 AdKDR-CDglyTK，能有效地感染 ECV304 和 HepG2 细胞，感染率依赖于重组腺病毒的 MOI。AdKDR-CDglyTK 感染的 ECV304 细胞对 GCV 和 5-FC 高度敏感，细胞存活率既依赖于前体药物的浓度，也依赖于重组腺病毒的 MOI。相反，HepG2 细胞无杀伤作用，两种前体药物联合应用比 GCV 或 5-FC 单独应用更有效。在有前体药物存在时，转基因 ECV304 细胞的生长受到抑制。
AdKDR-CDglyTK/双前体药物系统可能是抑制肿瘤血管生成的一种有效方法。
