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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 in vitro cytotoxicity of HSV tk / GCV suicide gene system on retinoblastoma (Rb) cells and the mechanism of bystander effect of herpes simplex virus thymidine kinase / gancyclovir (HSV tk / GCV) . Methods The plasmid pCMV / hytk IRES hrGFP was transfected into human retinoblastoma cell line HXO Rb44 using cationic liposomes. The transfection efficiency was observed under a fluorescence microscope. The positive cells transfected with HXO Rb44 / tk were screened by hygromycin. RT PCR to identify hytk gene in HXO Rb44 / tk cells in the integrated transcription results. The morphology and growth characteristics of Rb cells before and after transfection were compared. The killing effect of GCV on HXO Rb44 / tk cells and the cytotoxicity against mixed HXO Rb44 / tk and HXO Rb44 cells (“bystander effect”) were determined by MTT assay. The mechanism of bystander effect was studied through the experiment of supernatant shift. Results The transfection efficiency was about 20%. The 5 30bp hytk gene fragment was detected by RT PCR for HXO Rb44 / tk cell mRNA. The morphology and growth characteristics of Rb cells did not change significantly before and after transfection. HXO Rb44 / tk cells are highly sensitive to GCV and exhibit time and dose dependence. HSV tk / GCV system has obvious bystander effect on Rb cell killing, whereas GCV-treated HXO Rb44 / tk cell supernatant has no effect on HXO Rb44 cell growth. Conclusions HSV tk gene transfer combined with GCV may be an effective method for gene therapy of retinoblastoma. GCV metabolites may be bystander in this system through the cell gap junctions.