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...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.展开更多
文摘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.
