双自杀基因CD、HSV-tk共表达对肝门部胆管癌细胞杀伤作用实验研究

Killing effects on human hilar cholangiocarinoma cells with CD and HSV-tk double suicide genes coexpression

目的 :观察逆转录病毒介导的双自杀基因CD、HSV tk共表达对肝门部胆管癌细胞FRH的杀伤作用 ,探讨肝门部胆管癌的基因治疗方法。方法 :在脂质体Lipofectamine的介导下将含有双自杀基因的逆转录病毒载体PwzlneoCDglytk转导入包装细胞PA317,经G4 18筛选后培养产病毒的阳性克隆PA317/CD +tk细胞株 ,收集其病毒上清 ,转染FRH细胞 ,再次经G4 18筛选 ,获得稳定表达双自杀基因的FRH/CD +tk细胞株 ,用RT PCR检测双自杀基因的表达。给予前体药物 5 氟胞嘧啶 (5 flourocyto sine,5 Fc)和 /或无环鸟苷 (Ganciciovir,GCV)后 ,用MTT法测定转基因组及未转基因组FRH细胞的存活率。结果 :双自杀基因在FRH细胞中可稳定表达 ,联合使用 5 Fc和GCV对靶细胞增殖的杀伤作用及旁杀伤效应高于单独使用 5 Fc或GCV。结论 :逆转录病毒介导自杀基因可有效杀死肝门部胆管癌细胞 ,双自杀基因共表达较单一自杀基因有更强的抗肿瘤作用。

Objective:To investigate the different killing effects on human hilar cholangiocarinoma cells FRH with cytosine deaminase(CD) and herpes simplex virus thymidine kinase(HSV tk)double suicide genes coexpression compared with single gene mediated by retrovirus.To find a more efficient and low toxicity suicide gene therapy for hilar cholangiocarinoma.Methods:CD and HSV tk double suicide genes were transfected into PA317 cells using lipofectamine.The positive clones were picked out and cultured after G418 selected.The viral supernatant was collected.The FRH cells were infected with the virus containing the double suicide genes.After G418 selection,RT PCR was resorted to demonstrated the successful transcription of CD and HSV tk genes.The FRH/CD+tk and FRH cells in culture were respectively treated with 5 Fc and /or GCV.The cytoxicity efficacy was evaluated by microculture tetrajolium test (MTT) method.Results:The virus containing double suicide gene was produced in PA317 cells.Double suicide genes were stably expressed in RFH cells after being infected with the virus.The killing effect of combination 5 Fc with GCV on FRH/CD+tk cells is more effective than that of using 5 Fc or GCV alone.Conclusion:The CD+TK/5 Fc+GCV co expression system is more effective for killing effects on FRH cells than that of CD/5 Fc or tk/GCV system alone.