摘要
目的研究腺病毒介导的KDR-HSV-tk系统对血管内皮细胞的杀伤作用及旁杀效应。方法体外构建受KDR启动子和巨细胞病毒(CMV)启动子调控并可表达HSV-tk基因的Ad KDR-HSV-tk和Ad CMV-HSV-tk,经293细胞包装、扩增后,分别感染表达KDR的人脐静脉血管内皮细胞(HUVEC)和不表达KDR的人鼻咽癌细胞CNE-2,建立HUVEC/tk及CNE-2/tk,观察丙氧鸟苷(GCV)处理后HUVEC杀伤率及旁观者效应。结果受感染的HUVEC和CNE-2细胞中均有绿色荧光蛋白的表达。在感染复数(MOI)为100的条件下,GCV浓度由0增至50 mg/L时含Ad KDRHSV-tk的HUVEC和CNE-2细胞存活率从100%分别降至(23.1±3.9)%和(69.8±3.1)%(P<0.01);感染Ad CMV-HSV-tk的HUVEC和CNE-2细胞存活率从100%分别降至(13.9±2.9)%和(15.7±5.2)%(P<0.05);HUVEC/tk细胞对GCV的敏感性明显高于亲代HUVEC细胞,HUVEC+HUVEC/tk混合细胞的生存率随HUVEC/tk比例的增加而下降,说明Ad KDR-HSV-tk/GCV系统不但能杀伤转染tk基因的HUVEC,也能杀伤未导入转染tk基因的HUVEC细胞,存在明显的旁观者效应。结论腺病毒介导的KDR-HSV-tk/GCV系统对血管内皮细胞具有特异的杀伤作用及旁观者效应,为抗肿瘤血管靶向治疗奠定了基础。
Objective To study the killing and bystander effect of KDR-HSV-tk system study of adenovirus mediated vascular endothelial cells.Methods Ad KDR-HSV-tk and Ad CMV-HSV-tk controlled by KDR promoter and cytomegalovirus( CMV) promoter were constructed in vitro,and infected human umbilical vein endothelial cells( HUVEC) expressing KDR and human nasopharyngeal carcinoma cell line CNE-2 without KDR to establish HUVEC / tk and CNE-2 / tk respectively.The killing rate and bystander effect were observed after ganciclovir( GCV) treatment.Results The infected HUVEC and CNE-2 cells were found green fluorescent protein expression.At a multiplicity of infection( MOI) was 100,the concentration of GCV was increased from 0 to 50 mg / L Ad KDR-HSV-tk containing HUVEC and CNE-2 cell survival rate from 100% respectively( 23.1 ± 3.9) % and( 69.8 ± 3.1) %( P < 0.01);The survival rate from Ad CMV-HSV-tk infected HUVEC and CNE-2 cells were( 13.9 ± 2.9) % and( 15.7 ± 5.2) %;the sensitivity of HUVEC / tk cells to GCV was significantly higher than that in parental HUVEC cells,the survival rate of HUVEC + HUVEC / tk hybrid cells was increased with the percentage of HUVEC / tk cells decreasing,indicating that the Ad KDR-HSV-tk / GCV system didnot kill tk+cells,and also killed without tk gene into cells.Conclusions The KDR-HSV-tk / GCV system mediated by adenovirus has killing and bystander effect on vascular endothelial cell specific,as anti-angiogenic target lays the experimental basis to the treatment.
出处
《中国老年学杂志》
CAS
CSCD
北大核心
2015年第15期4131-4133,共3页
Chinese Journal of Gerontology
基金
国家自然科学基金资助项目(81350022)
