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  

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

作　　者：朱欠元[1] 李宝金[2] 郭剑[1] 毛慕华[1] 

机构地区：[1]井冈山学院附属医院耳鼻喉科,江西吉安343000 [2]北京大学深圳医院肝胆胰外科,广东深圳518036

出　　处：《中国临床医学》2006年第4期522-524,共3页Chinese Journal of Clinical Medicine

基　　金：江西省卫生厅科技资助项目(项目编号20052041)

摘　　要：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混合细胞还随丙氧鸟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／丙氧鸟苷系统对血管内皮细胞具有明显的杀伤及旁观者效应。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 detection. The HUVEC/tk cells showed significantly higher sensitivity to GCV than parental HUVEC cells. HUVEC/tk and HUVEC-F HUVEC/tk mixed cells were obviously suppressed with the gradutional increase of GCV concentration. And the survival rate of HUVEC-F 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.

关 键 词：KDR启动子 单纯疱疹病毒胸苷激酶 旁观者效应 血管内皮 

分 类 号：R645.2[医药卫生—外科学]