CAJ | 학술논문

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AdKDR－HSV－tk和AdCMV－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 AdKDR－HSV－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AdCMV－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AdKDR－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。
목적：연구선병독개도적KDR－HSV－tk계통대혈관내피세포적살상작용급방살효응。방법체외구건수 KDR계동자화거세포병독（ CMV）계동자조공병가표체HSV－tk기인적AdKDR－HSV－tk화AdCMV－HSV－tk，경293세포포장、확증후，분별감염표체KDR적인제정맥혈관내피세포（HUVEC）화불표체KDR적인비인암세포CNE－2，건립HUVEC／tk급CNE－2／tk，관찰병양조감（GCV）처리후 HUVEC 살상솔급방관자효응。결과수감염적HUVEC화CNE－2세포중균유록색형광단백적표체。재감염복수（ MOI）위100적조건하，GCV농도유0증지50 mg／L 시함 AdKDR－HSV－tk적HUVEC화CNE－2세포존활솔종100％분별강지（23．1±3．9）％화（69．8±3．1）％（P＜0．01）；감염AdCMV－HSV－tk적HUVEC화 CNE－2세포존활솔종100％분별강지（13．9±2．9）％화（15．7±5．2）％（P＜0．05）；HUVEC／tk세포대GCV적민감성명현고우친대HUVEC세포， HUVEC＋HUVEC／tk혼합세포적생존솔수HUVEC／tk비례적증가이하강，설명AdKDR－HSV－tk／GCV계통불단능살상전염tk기인적HUVEC，야능살상미도입전염tk기인적HUVEC세포，존재명현적방관자효응。결론선병독개도적KDR－HSV－tk／GCV계통대혈관내피세포구유특이적살상작용급방관자효응，위항종류혈관파향치료전정료기출。
Objective To study the killing and bystander effect of KDR-HSV-tk system study of adenovirus mediated vascular endo-thelial cells.Methods AdKDR-HSV-tk and AdCMV-HSV-tk controlled by KDR promoter and cytomegalovirus (CMV) promoter were con-structed 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 ganci-clovir(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 AdKDR-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 AdCMV-HSV-tk infected HU-VEC 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 de-creasing,indicating that the AdKDR-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 .