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研究(英文)

Antitumor effects of HSV-tk/GCV suicide gene system for retinoblastoma in vitro

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 应用阳离子脂质体将pCMV/hytk IRES hrGFP质粒导入人视网膜母细胞瘤株HXO Rb44细胞 ,荧光显微镜下观察转染效果 ,并用潮霉素筛选出转染阳性细胞HXO Rb44/tk。RT PCR鉴定hytk基因在HXO Rb44/tk细胞中的整合转录结果。比较转染前后Rb细胞的形态和生长特性。通过MTT法检测GCV对HXO Rb44/tk细胞的杀伤作用以及对不同比例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 转染效率约为2 0 %。HXO Rb44/tk细胞mRNA经RT PCR可检测出 5 30bp的hytk基因片段。转染前后Rb细胞的形态和生长特性无明显改变。HXO Rb44/tk细胞对GCV高度敏感 ,并表现出时间和剂量的依赖性。HSV tk/GCV系统对Rb细胞的杀伤过程中存在明显的旁观者效应 ,而GCV作用的HXO Rb44/tk细胞上清对HXO Rb44细胞生长无影响。结论HSV tk基因转移联合GCV治疗可作为视网膜母细胞瘤基因治疗的一种有效方法 ,GCV代谢产物通过细胞间缝隙连接进行转运可能是此系统旁观者?

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.A fluorescence microscope was used to detect the transduction effeciency. The positive cell clones were selected by hygromycin and were named HXO Rb 44 /tk. RT PCR was resorted to demonstrate the sucessful transduction and 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 determined the killing effect of GCV on HXO Rb 44 /tk and 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 The transduction effeciency was 20%. 530bp 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 .HXO Rb 44 /tk was more sensitive to GCV than was HXO Rb 44 .The cytotoxicity of HXO Rb 44 /tk was dose and time dependent. An obvious 'bystander effect' was seen even with low proportions of HXO Rb 44 /tk, but this effect disappeared when transferring GCV containing supernatant of HSV tk positive cells to the negative cells.Conclusions 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 retinoblastoma, the 'bystander effect' in HSV tk/GCV mediated gene therapy occurs by transfer of GCV metabolite from cell to cell through gap junction.