腺病毒介导的KDR启动子-胸苷激酶系统对血管内皮细胞旁观者效应的实验研究

目的：研究腺病毒介导的血管内皮因子受体（KDR）启动子-胸苷激酶系统对血管内皮细胞的旁观者效应。方法：采用新型腺病毒载体AdEasy系统，构建由KDR启动子调控的、腺病毒介导的、单纯疱疹病毒胸苷酸激酶基因系统（AdK—DR—HSv-tk），在人胚胎肾细胞系293细胞中包装、扩增后，体外感染表达KDR的人脐静脉血管内皮细胞（HUVEC），建立HUVEC／tk转化细胞株。观察丙氧鸟苷（GCV）对HUVEC、HUVEC／tk细胞的存活率及旁观者效应。结果：从病毒滴度及PCR检查，证实tk基因随病毒的感染已成功地导入293细胞及HUVEC细胞。HUVEC／tk细胞对丙氧鸟苷的敏感性显著高于亲代HUVEC细胞。HUVEC／tk及HUVEC＋HUVEC／tk混合细胞还随丙氧乌苷浓度的增加受到明显的抑制。HUVEC＋HUVEC／tk混合细胞的存活率，随HUVEC／tk细胞比例的增加而降低，说明腺病毒介导的KDR启动子-单纯疱疹病毒胸苷激酶／丙氧鸟苷系统不但能杀伤tk^＋细胞，也能杀伤未导入tk的细胞，存在明显的旁观者效应。结论：腺病毒介导的KDR启动子单纯疱疹病毒胸苷激酶／丙氧乌苷系统对血管内皮细胞具有明显的杀伤及旁观者效应。

The Bystander Effect of Adenoviral Mediated Gene Transfer of Herpes Simplex Virus Thymidine Kinase under the Control of KDR Promote on Human Vascular Endothelial Cells

Objective: To investigate the bystander effect of the adenoviral vector containing KDR promoter for the HSV- tk gene on the vascular endothelial cells. Methods:By using Adeasy system, recombinant adenoviral plasmid containing KDR promoter-controlled HSV-tk gene (AdKDR-HSV-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 establish transfection cell lines. The effect of GCV on survival rate and bystander effect of HUVEC and HUVEC/tk cells was observed by cell culture and MTT test. Results:Transduction of tk gene into 293 cells and HUVEC cell lines was successful and validated by viral titer and PCR detectioa The HUVEC/tk cells showed significantly higher sensitivity to GCV than parental HUVEC cells. HUVEC/tk and HUVEC+ HUVEC/tk mixed cells were obviously suppressed with the gradutional increase of GCV concentration.And the survival rate of HUVEC+HUVEC/tk mixed cells decreased with the increase of HUVEC/tk cells. It implied that tk/GCV system could kill not only tk cells but also untransdmixed cells,thus verifying the presence of bystander effect. Conclusion:The above results demonstrate adenovirus-mediated transfection of KDR promoter-HSV-tk gene has obvious killing and bystander effect on vascular endothelial cells with treatment of GCV.