双自杀基因慢病毒转移系统的建立及其对T淋巴细胞的体外杀伤作用

目的利用分子生物学方法构建CD和豫双自杀基因慢病毒转移载体。方法将慢病毒基因转移系统中的3种成分质粒（包膜质粒、包装质粒及目的基因转移质粒）用脂质体共转染人病毒包装细胞293T，荧光显微镜下观察；透射电镜下观察病毒颗粒；大量收集病毒上清，浓缩后用之感染T淋巴细胞，荧光显微镜下观察。结果荧光显微镜观察到对照组大量绿色荧光蛋白（GFP）；透射电镜证实有大量病毒颗粒存在。单独使用前体药物5-氟脲嘧啶（5-FC）和／或无环鸟苷（GCV）后，细胞的存活率与未转染T淋巴细胞比较，差异显著（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）。结论慢病毒基因转移系统可使双自杀基因发挥强大的杀伤作用，为一种有效的基因转移系统。

The experimental study on T lymphocytes killing effects of lentivirus-mediated double suicide gene system

Objective Double suicide gene lentivirus vector was successfully constructed using molecular method.Methods Three plasmids of lentivirus gene transfer vector system were effectively transferred into packaging cell line 293T using lipofectine method.So much green fluorescence was observed in fluorescence microscopy.A lot of lentivirus particles were identified in electronic microscopy.High titer lentivirus was harvested and concentrated.The T lymphocytes were infected with concentrated virus containing the double suicide genes.Double suicide genes were stably expressed in T lymphocytes after being infected with concentrated virus using fluorescence microscopy and RT-PCR.Extraneous gene integration and expression were confirmed by fluorescence microscopy and RT-PCR.Results The cytotoxicity to these transgenic cells treated with 5-FC and GCV was measured by MTT assays and flow cytometry(FCM).The killing effects of Combination 5-FC with GCV on T lymphocytes was more effective than that of using 5-FC or GCV alone(P<0.01). Lentivirus-mediated gene transfer system can transfer CD and TK double suicide genes into T cells with affording strong killing effects when given 5-FC or GCV.