慢病毒介导的双自杀基因对T淋巴细胞杀伤作用的实验研究

Killing effect of double suicide genes on T lymphocytes mediated by lentivirus

目的:探讨慢病毒介导的双自杀基因对T淋巴细胞的杀伤作用。方法:在脂质体介导下将慢病毒包装系统的包装结构基因pCMVΔ8.2、包膜基因pCMV.VSVG、目的基因pHR'CS.GFP或pHR'CS.CDglytk导入病毒包装细胞293T,包装成病毒后,收集病毒上清,浓缩后转染T淋巴细胞,荧光显微镜及RT-PCR检测基因的整合及表达。给予前体药物5-氟胞嘧啶(5-FC)和/或无环鸟苷(GCV)后,MTT法测定细胞的存活率,流式细胞仪检测细胞的凋亡率。结果:慢病毒的3种质粒可高效转染入293T细胞。对照组荧光显微镜下观察可见大量的绿色荧光,透射电镜下观察可见许多病毒颗粒。慢病毒介导的双自杀基因在T淋巴细胞中高效、稳定表达,单独使用GCV或5-FC对T细胞/CD+tk的存活率与未转染T细胞比较明显降低(P<0.01),与单独使用5-FC或GCV比较,联合使用5-FC和GCV时T细胞的存活率明显降低(P<0.01)。结论:慢病毒介导的双自杀基因可高效稳定转染T淋巴细胞,双自杀基因系统较单一自杀基因系统(CD/5-FC或HSV-tk/GCV)对T淋巴细胞的杀伤具有明显增强作用。

Objective:To explore the killing effect of double suicide genes mediated by lentivirus on T lymphocytes.Methods :The three plasmids expressing lentivirus,packaging plasmid pCMVΔ8.2,envelope plasmid pCMV.VSVG and target plasmid(pHR'CS.GFP as control group,pHR'CS.CDglytk as experiment group)were packaged into virus packaging cell line293T using lipo-fectine method.The virus-producing cell supernatant was harvested and concentrated.The T lympho-cytes were infected with the concentrated virus.The gene integration and expression were confirmed by fluorescence microscopy and RT-PCR.After prodrug GCV or /and5-FC were administered,MTT method was used to detect the growth rate of T lymphocytes and flow cytometry(FCM)was used to evaluate the apoptosis rate of T lymphocytes.Results:The three plasmids were effectively transferred into293T cells.So much green fluorescence was observed through fluorescence microscopy and a lot of virus particles were observed through transmission electronic microscopy.Double suicide genes mediated by lentivirus were effectively and stably expressed in T lymphocytes.The growth rate of T lymphocytes given GCV or5-FC was apparently lower than that of untransfected T lymphocytes(P<0.01).When GCV and5-FC were used jointly,the growth rate of T lymphocytes was apparently lower than that ofthe group given GCV or5-FC alone(P<0.01).Conclusion:Double suicide genes mediated by lentiviral vector could transfect T lymphocytes effectively and stably.The double suicide gene system enhanced killing effect on T lymphocytes more remarkably than CD/5FC or HSV-tk/GCV system alone.