双自杀基因对Raji淋巴瘤细胞杀伤的增强作用

ENHANCED KILLING EFFECT OF DOUBLE SUICIDE GENES TRANSFERRED INTO Raji LYMPHOMA CELLS BY RETROVIRAL VECTOR

目的 :观察反转录病毒介导的双自杀基因对 Raji淋巴瘤细胞的杀伤增强作用 ,探讨淋巴瘤的基因治疗方法。方法 :通过脂质体将含有双自杀基因的反转录病毒载体 p WZL neo CDglytk导入病毒包装细胞 PA317,经 G4 18筛选后大量培养产病毒的阳性克隆 PA317/ CD+ tk细胞株 ,收集病毒上清 ,浓缩后转染 Raji淋巴瘤细胞 ,再次经 G4 18筛选 ,获得稳定表达双自杀基因的 Raji/ 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 - flourocytosine,5 - FC)和 /或无环鸟苷(Ganciciovir,GCV)后 ,MTT法测定细胞的存活率及 CDglytk双自杀基因对 Raji细胞的杀伤作用。结果 :双自杀基因在 Raji细胞中可稳定表达 ,联合使用 5 - FC和 GCV时 Raji细胞的存活率 (13.83% )明显低于单独使用 GCV (5 0 .6 5 % )或 5 - FC(5 7.6 8% )时 ,各组比差异具有显著性 (P<0 .0 1)。结论 :反转录病毒介导双自杀基因对

Objective:To investigate the different killing effects on lymphoma Raji cells with cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV tk) double suicide genes coexpressed compared with single gene mediated by retrovirus. To find a more efficient and low toxicity suicide gene therapy for lymphoma.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 and concentrated.The lymphoma cells were infected with the concentrated virus containing the double suicide genes.After G418 selection, RT PCR was resorted to demonstrate the successful transcription of CD and HSV tk genes.The Raji/CD + tk and Raji 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 and efficiently expressed in Raji cells after being infected with the provirus.Exposure to the matching prodrugs (GCV and 5 FC) showed decreased cell growth rate (13.83 %) compared with exposure to 5 FC (57.68 %) or GCV (50.65 %) alone.There are remarkably significant between the groups.Conclusion:The double suicide gene system mediated by retroviral vector enhanced killing effect remarkably on lymphnoma cells than CD/5 FC or tk/GCV system alone.The killing effect of combination 5 FC with GCV on Raji/CD + tk cells is more effective than that of using 5 FC or GCV alone. The CD + tk /5 FC+GCV co expression system is more effective for killing effect on lymphoma cells than that of CD/SFC or TK/GCV system alone.