HSV-tk/GCV自杀基因系统对视网膜母细胞瘤的体外抗肿瘤效应  

Antitumor Effects of HSV-tk/GCV Suicide Gene System for Retinoblastoma in vitro

作　　者：李涛[1] 张虹[1] 王骞[2] 梁峰[3] 李伟[3] 李贵刚[1] 卢运萍[4] 

机构地区：[1]华中科技大学同济医学院附属同济医院眼科,武汉430030 [2]新加坡国立大学生物系,新加坡119260 [3]武汉大学化学系,武汉430072 [4]华中科技大学同济医学院附属同济医院肿瘤生物医学中心,武汉430030

出　　处：《华中科技大学学报（医学版）》2003年第5期533-537,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

摘　　要：目的　研究单纯疱疹病毒胸腺嘧啶核苷激酶 /更昔洛韦 (herpessimplexvirusthymidinekinase /gancyclovir,HSV tk/GCV)自杀基因系统对视网膜母细胞瘤 (retinoblastoma ,Rb)细胞的体外杀伤作用以及旁观者效应发生的机制。方法　应用脂质体将pCMV/hytk IRES hrGFP质粒导入HXO Rb44细胞株。用潮霉素筛选出阳性细胞克隆并命名为HXO Rb44/tk。RT PCR鉴定hytk基因在HXO Rb44/tk细胞中的转录结果。比较HXO Rb44和HXO Rb44/tk细胞的形态特征及生长特性。通过MTT法检测GCV对不同比例HXO Rb44/tk和HXO Rb44混合细胞的杀伤作用 (“旁观者效应”)。并通过上清移换实验研究旁观者效应发生的机制。结果　HXO Rb44/tk细胞经RT PCR可检测出 5 30bp的hytk基因片段。HXO Rb44/tk和HXO Rb44细胞的形态特征及生长特性无明显差异。HXO Rb44/tk细胞仅占很低比例时即可观察到明显的旁观者效应 ,而GCV作用的HXO Rb44/tk细胞上清对HXO Rb44细胞无杀伤作用。结论　HSV tk基因转移联合GCV治疗可作为Rb基因治疗的一种有效方法 。Objective To investigate the killing effect of herpes simplex virus thymidine kinase/gancyclovir (HSV tk/GCV) suicide gene therapy system on retinoblastoma (Rb) cells and the mechanism of bystander effect. Methods By using liposome, pCMV/hytk IRES hrGFP plasmid was transferred into HXO Rb 44 cells. The positive cell clones were selected by hygromycin and were named as HXO Rb 44 /tk. RT PCR were resorted to demonstrate the transcription of hytk gene in the HXO Rb 44 /tk cells. The morphologic features and growth patterns of HXO Rb 44 /tk were compared with those of HXO Rb 44 . Then MTT assay was used to determine the killing effect of GCV on the mixture of HXO Rb 44 /tk and HXO Rb 44 in different ratios ('bystander effect'). The mechanism of bystander effect was studied by the experiment of supernatant shifting. Results 530 bp hytk gene strand was seen through HXO Rb 44 /tk RT PCR. There were no differences in the morphologic features or the growth patterns between HXO Rb 44 /tk and HXO Rb 44 . An obvious 'bystander effect' was seen even with low proportions of HXO Rb 44 /tk, but there was no killing effect of GCV containing supernatant of HXO Rb 44 /tk on HXO Rb 44 . Conclusion The transfer of the HSV tk gene into Rb cells followed by the administration of GCV could serve as a model for gene therapy for Rb and the bystander effect occurs by transfer of GCV metabolite from cell to cell through gap junction.

关 键 词：HSV—tk/GCV 自杀基因系统 视网膜母细胞瘤 体外抗肿瘤效应 基因疗法 胸腺嘧啶核苷激酶 

分 类 号：R739.7[医药卫生—肿瘤]