目的:探讨缺氧条件下,缺氧反应元件启动子-胸苷激酶/丙氧鸟苷(HRE-TK/GCV)系统对骨肉瘤细胞系MG63靶向性杀伤作用。方法:构建由HRE启动子驱动的含潮霉素磷酸转移酶-单纯疱疹病毒胸腺嘧啶激酶(Hygromycinphosphotransferase-thymidinekinase,HyTK)融合基因的真核表达质粒pBI-HRE-HyTK,并将其转染入骨肉瘤细胞系MG63。应用PCR和RT-PCR方法检测TK基因的整合及表达;用MTT法和混合共培养实验分别检测转基因细胞在缺氧或不缺氧状态下对GCV的敏感性以及HRE-TK/GCV系统的“旁观者效应”;流式细胞仪检测细胞周期改变及电镜观察细胞超微结构变化探讨其作用机制。结果:成功构建了真核表达质粒pBI-HRE-HyTK,得到转基因细胞系MG63TK。检测到MG63TK细胞内TK基因的DNA和mRNA。MG63TK细胞在不缺氧状态下对GCV不敏感,当GCV浓度达50μg/ml时,仅30%左右细胞被杀死;而缺氧状态下,GCV浓度1μg/ml时,50%左右细胞被杀死,GCV浓度50μg/ml时,90%以上细胞被杀死。混合共培养实验中,缺氧状态下,HRE-TK/GCV系统旁观者效应明显增强,MG63细胞存活率显著低于不缺氧状态下。缺氧和GCV共同作用使转基因细胞DNA合成抑制,细胞周期阻滞于G0G1期,细胞发生凋亡和坏死。结论:缺氧可以增强HRE-TK/GCV系统对体外培养骨肉瘤细胞系选择性的杀伤作用? OBJECTIVE: To investigate the targeted killing of osteosarcoma cell line MG63 by hypoxia-responsive promoter-thymidine kinase / gonadoside (HRE-TK / GCV) system in hypoxia. Methods: The eukaryotic expression plasmid pBI-HRE-HyTK containing hygromycin phosphotransferase-hygromycinphosphotransferase-thymidine kinase (HyTK) fusion gene driven by HRE promoter was constructed and transfected into osteosarcoma Cell line MG63. The integration and expression of TK gene were detected by PCR and RT-PCR. MTT assay and co-culture assay were used to detect the sensitivity of transgenic cells to GCV in hypoxia and not hypoxia, respectively. Bystander effect ". The changes of cell cycle were detected by flow cytometry and the ultrastructural changes of cells were observed by electron microscope. Results: The eukaryotic expression plasmid pBI-HRE-HyTK was successfully constructed and the transgenic cell line MG63TK was obtained. The DNA and mRNA of TK gene in MG63TK cells were detected. In the absence of hypoxia, MG63TK cells were not sensitive to GCV. Only 30% of cells were killed when the concentration of GCV was 50μg / ml. In hypoxia, 50% of cells were killed when the concentration of GCV was 1μg / ml When dead, at a GCV concentration of 50 μg / ml, more than 90% of the cells were killed. In co-culture experiment, the bystander effect of HRE-TK / GCV system was significantly increased under hypoxia condition, and the viability of MG63 cells was significantly lower than that under hypoxia condition. Hypoxia and GCV combined to inhibit the synthesis of DNA in transgenic cells, cell cycle arrest in G0G1 phase, cell apoptosis and necrosis. Conclusion: Hypoxia can enhance the selective killing effect of HRE-TK / GCV system on osteosarcoma cell lines in vitro.