慢病毒携带的双自杀基因对淋巴瘤细胞杀伤作用的实验研究

Killing Effect of Double Suicide Gen es Mediated by Lentivirus on Lymphoma Cells

背景与目的:慢病毒载体具有可感染非分裂细胞、目的基因整合至靶细胞基因组长期表达、免疫原性低等优点,适于体内基因治疗。本研究探讨慢病毒介导的双自杀基因对淋巴瘤细胞Raji的杀伤作用。方法:将表达慢病毒的3种质粒,即包装结构基因质粒pCMVΔ8.2、包膜基因质粒pCMV.VSVG、目的基因质粒pHR'CS.GFP或pHR'CS.CDglytk分两组(pHR'CS.Cdglytk为实验组、pHR'CS.GFP为对照组),经脂质体导入病毒包装细胞293T,包装成病毒后,收集病毒上清,浓缩后转染Raji细胞,用荧光显微镜及RT-PCR检测基因的表达。给予前体药物5-氟胞嘧啶(5-FC)和/或无环鸟苷(GCV),用MTT法测定Raji细胞的生长抑制率,检测CD和HSV-tk双自杀基因对Raji细胞的作用。结果:表达慢病毒的3种质粒可高效转染入293T细胞。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富集的病毒颗粒。慢病毒介导的双自杀基因在Raji细胞中高效、稳定表达。单独使用GCV或5-FC对细胞的生长抑制率分别为51%、50%,与未转染组Raji细胞比较,差异有显著性(P<0.01);联合使用5-FC和GCV对细胞的生长抑制率为73%,明显高于单独使用5-FC或GCV(P<0.01)。结论:慢病毒介导的双自杀基因可高效稳定转染淋巴瘤细胞;双自杀基因系统较单一自杀基因系统(CD/5-FC或HSVtk/GCV)对淋巴瘤细?

BACKGROUND &OBJECTIVE:The lentiviral vectors can integ rate interest g enes into g enom e of the targ et cells that allow for st able transg enic expression even in non-d ividing cells without evoking an imm une response of the host.All the feature s have promised them to be used in vivo g ene therapy.This study was desig ned to explore the killing effect of dou ble suicide g enes mediated by lentiviru s on lymphoma cells(Raji).METHODS :The three plasmids expressed lentiviru s ,packag ing plasmid pCMV 8.2,envel ope plasmid pCMV.VSVG and targ et plasmi d(pHR'CS.GFP as control g roup,pHR' CS.CDg lytk as experiment g roup)were packag ed into 293T cells using lipofectine method.Supernatant wa s harvested and concentrated.The Ra ji cells were infected with the concentrated virus.The g ene integ ration and expr ession were confirmed by fluorescence micr oscopy and RT-PCR.After prodrug GCV or /and 5-FC administration,MTT met hod was used to detect the g rowth inhibition rate(GIR)of Raji cells for evaluating the kill ing effect of CD and HSV-tk double suicide g enes on Raji cells.RESULTS :The three plasmids were effectively transferred into 293T c ells.Green fluorescence on the cell was observed throug h fluorescence micr oscopy and a lot of virus particles we re observed throug h transmission elec tronic microscopy.Double suicide g enes mediated by lentivirus were effectively and stably expressed in Raji cel ls.The GIR of Raji cells using GCV or 5-FC was 51%or 50%,respectively,and it was apparently hig her than that of untra nsfected cells(P<0.01).When using GCV and 5-FC tog ether,the GIR was 73%,wh ich was apparently hig her than that o f g roup using GCV or 5-FC alone(P<0.01).CONCLUSION:Double suicide g enes mediated by lentiviral vector could transfect lymphoma cells effe ctively and stably.The double suicide g ene syst em enhanced killing effect remarkab ly on lymphoma cells than CD /5FC or HSV-tk/GCV system alone.