目的观察双自杀基因的共表达与单自杀基因单独表达对癌细胞的杀伤作用。方法分别构建了以CMV为启动子，含单纯疱疹病毒胸苷激酶(HSV-TK)和/或大肠杆菌胞嘧啶脱氨酶(Ecoli.CD)单、双自杀基因的真核表达载体。在脂质体介导下将基因导入细胞，经G418筛选出稳定表达的克隆。用PCR、半定量RT-PCR检测各组基因的整合及表达。给予前体药物5-氟胞嘧啶(5-flourocytosine,5-Fc)和/或无环鸟苷(Gauciclovir,GCV)后，用MTT法测定各转基因组细胞的存活率。结果单、双自杀基因均在GLC-82细胞中稳定表达，双基因转染组对细胞增殖的杀伤及旁杀伤效应高于单基因组。结论 TK+CD/5-Fc+GCV的双基因共表达体系较CD/5-Fc或TK/GCV单基因体系对细胞具有更强的杀伤作用。%Objective To investigate the different killing effect to human pulmonary adenocarcinoma cell line cells GLC-82 with coexpressed double suicide genes compared with single gene. Methods Recombinant expression vectors containing CD (cytosine deaminase) and/or TK(thymidine kinase) gene under CMV promoter were constructed successfully. The vectors were transfected to GLC-82 tumor cell lines by use of lipofectamine. The clones were picked out after G418 selection. Extraneous gene integration and expression were confirmed by PCR and semi-quantitative RT-PCR. The cytotoxicity to these transgenic cells under treatment with 5-Fc and GCV were measured by MT1 assays. Results Double and single suicide gene transfer were both stably expressed in GLC-82 cells . The cytotoxic effects of co-expressed TK-CD genes were superior than that of the single gene. Conclusion The CD + TK/5-Fc + GCV coexpression system is more effective for killing effect of tumor cells than CD/5-Fc or TK/GCV system alone.
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