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本工作在血管内皮细胞中分别建立了HSV-tk/GCV和EC-cd/5-FC自杀基因系统,用于抑制实体肿瘤中血管生成的研究.构建含自杀基因的重组腺病毒AdCMVtk和AdCMVcd,滴度均1×10~(12)pfu/ml.病毒感染小鼠血管内皮细胞lGll(MOI=100),并鉴定自杀基因的表达,测定原药对它们的杀伤.生长抑制试验表明,GCV对lGll/Ad-tk细胞杀伤的IG_(50)为0.2μmol/ L,5-FC对lGll/Ad-cd细胞杀伤的IC_(50)为20μmol/L.电镜下显示被原药杀伤的血管内皮细胞有明显的细胞凋亡现象.同时混合细胞实验证明两系统均存在明显的旁杀伤效应.从而在体外水平证明了HSV-tk/GCV和EC-cd/5-FC自杀基因系统杀伤增生血管内皮细胞的有效性.
In this work, HSV-tk / GCV and EC-cd / 5-FC suicide gene systems were established in vascular endothelial cells to inhibit angiogenesis in solid tumors.Construct the recombinant adenovirus AdCMVtk and AdCMVcd containing suicide gene, The virus titer was 1 × 10 ~ (12) pfu / ml. The virus infected mouse vascular endothelial cells lGll (MOI = 100), and identified suicide gene expression, determination of the original drug to kill them. Growth inhibition test showed that, The IC50 of lGll / Ad-tk cell killing was 0.2μmol / L and that of 5-FC on lGll / Ad-cd cell killing was 20μmol / L. Electron microscopy showed that the drug-killed vascular endothelial cells There was obvious phenomenon of cell apoptosis.At the same time mixed cell experiments showed that both systems have significant para-lethal effect.Thus it was demonstrated in vitro that HSV-tk / GCV and EC-cd / 5-FC suicide gene systems kill proliferating vascular endothelial cells Effectiveness.