【摘要】 目的构建含以KDR为启动子的HSV-tk重组腺病毒,体外评估其对血管内皮细胞的特异性杀伤效能。方法采用pAdeasy系统,构建受KDR启动子或巨细胞病毒(CMV)启动子调控并可表达HSV-tk基因的AdKDR-tk和AdCMV-tk,在293细胞中包装、扩增后,体外分别感染表达KDR的人脐静脉血管内皮细胞系(HUVEC)和不表达KDR的肝癌细胞系HepG2,用丙氧鸟苷(GCV)处理受染细胞,并以MTT法检测其细胞增殖情况。结果病毒滴度均为1×1010pfu/mL。在感染复数(MOI)为100的条件下,当细胞培养液中加入的GCV浓度由0增至50μg/mL时,感染含AdKDR-tk的HUVEC细胞和HepG2细胞其存活率由100%分别下降至(28.94±5.67)%和(75.45±2.91)%(P<0.01),而感染含AdCMV-tk的HUVEC细胞和HepG2细胞其存活率分别下降至(17.56±2.48)%和(23.15±5.72)%(P>0.05)。结论KDR启动子介导的HSV-tk具有特异性杀伤血管内皮细胞的作用。更多还原

【Abstract】 Objective To construction of recombinant adenovirus containing KDR promoter-HSV-tk suicide gene by using a pAdeasy system and evaluate its specific killing effect on vascular endothelial cells. Methods By using pAdeasy system, recombinant adenoviral plasmid containing KDR or cytomegalovirus (CMV) promoter-controlled HSV-tk gene (AdKDR-tk and AdCMV-tk) was constructed. After packaging and amplification in 293 cells, the virus was used to infect KDR-expressed human umbilical venous endothelial cells (HUVEC) and KDR-unexpressed HepG2. Following administration of ganciclovir(GCV), the survival rate of gene-transfected HUVEC and HepG2 was evaluated by using MTT method. Results The pAdeasy System produced a higher titer of the recombinant adenovirus (1×1010pfu/ml). Under infection index of 100, with increasing GCV concentration from 0 up to 50ug/ml, the survival rate of AdKDR-tk-transfected HUVEC and HepG2 decreased from 100% to (28.94±5.67)% and(75.45±2.91)%, respectively (P<0.01), while the survival rate of AdCMV-tk-transfected HUVEC and HepG2 declined from 100% to (17.56±2.48)% and (23.15±5.72)%, respectively (P>0.05). Conclusions KDR promoter-HSV-tk gene possess a specific killing effect on vascular endothelial cells treated with GCV.更多还原