Huang Zong-hai, Yang Wen-yu, Gong Xiao-wei, Qian Yong, Che Xiao-yan
Department of Surgery, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, China.
Di Yi Jun Yi Da Xue Xue Bao. 2004 Feb;24(2):139-43.
To study the killing effect of adenovirus-mediated double suicide gene under the regulation of kinase domain-containing receptor (KDR) promoter on human umbilical vein endothelial cells (HUVECs).
The sequences of human KDR promoter gene, CD gene and TK gene were amplified by PCR, and the plasmid pKDR-CDglyTK was constructed. A two-step transformation protocol was employed for the construction of a recombinant adenoviral plasmid pAdKDR-CDglyTK that was transfected into 293 packaging cells to further multiply and purify the adenovirus. HUVECs were infected by the resultant recombinant adenovirus of different multiplicities of infection (MOI), and the infection rate was measured by observing the expression of green fluorescence protein (GFP). The infected cells were cultured in the culture media containing ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the killing effects were evaluated.
Recombinant adenovirus AdKDR-CDglyTK were successfully constructed, which could efficiently infect HUVEC cells, with the infection rate associated with the MOI of the recombinant adenovirus. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of the recombinant adenovirus. The killing effect of the two produrgs used in combination was much stronger than that of exclusive use of GCV or 5-FC.
Prodrug/KDR-CdglyTK system is effective in killing HUVEC cells, and its killing effect is correlated with the concentration of the prodrugs and the MOI of the recombinant adenovirus. Combination of the two prodrugs produces stronger killing effect on the cells.
研究含激酶结构域受体（KDR）启动子调控下的腺病毒介导双自杀基因对人脐静脉内皮细胞（HUVECs）的杀伤作用。
采用聚合酶链反应（PCR）扩增人KDR启动子基因、CD基因和TK基因序列，构建质粒pKDR - CDglyTK。采用两步转化法构建重组腺病毒质粒pAdKDR - CDglyTK，将其转染至293包装细胞中进一步扩增和纯化腺病毒。用不同感染复数（MOI）的重组腺病毒感染HUVECs，通过观察绿色荧光蛋白（GFP）表达测定感染率。将感染后的细胞在含不同浓度更昔洛韦（GCV）和/或5 - 氟胞嘧啶（5 - FC）的培养基中培养，评估杀伤效果。
成功构建重组腺病毒AdKDR - CDglyTK，其能高效感染HUVEC细胞，感染率与重组腺病毒的MOI相关。感染AdKDR - CDglyTK的HUVEC细胞对前体药物高度敏感，其存活率与前体药物浓度及重组腺病毒的MOI均相关。两种前体药物联合使用的杀伤效果远强于单独使用GCV或5 - FC。
前体药物/KDR - CdglyTK系统对HUVEC细胞有杀伤作用，其杀伤效果与前体药物浓度及重组腺病毒的MOI相关。两种前体药物联合对细胞产生更强的杀伤作用。
