目的:探讨运用增强子上调肿瘤特异性启动子h TERT转录活性后,调控自杀融合基因CDTK对人肝癌细胞系Bel-7402的体内外杀伤作用。方法:将CMV增强子、h TERT启动子及CDTK自杀融合基因克隆入核糖体基因区打靶载体p Hrn,构建p Hr-Ce Tp CDTK-GFP治疗载体并转染Bel-7402细胞,经RT-PCR、HPLC、MTT检测CDTK基因的表达和对肝癌细胞的体外杀伤效果。再将Bel-7402细胞接种到裸鼠皮下致瘤,以肿瘤杀伤载体p Hr-Ce Tp CDTK-GFP进行瘤内转染并检测其抑瘤效果,此外将转染后的细胞接种致瘤,检测其成瘤时间及肿瘤生长曲线等,从两方面检测其体内杀伤效果。结果:成功构建了肿瘤特异性治疗载体,体外转染肝癌细胞后,经RTPCR、HPLC证明CDTK基因能在肝癌中表达,且治疗载体在体外对肝癌细胞有明显毒性。动物实验结果发现裸鼠致瘤后治疗组血清中5-Fu浓度为7.694μg/ml,治疗组肿瘤体积与对照组相比减小6.5倍。而细胞转染治疗载体后致瘤,治疗组成瘤时间比对照组晚8天,且治疗组裸鼠的平均生存期较对照组延长16天。结论:p Hr-Ce Tp CDTK-GFP载体能在体内外高效靶向杀伤肝癌细胞,为肝癌基因治疗提供了一种新的途径。 OBJECTIVE: To investigate the in vivo and in vitro cytotoxicity of suicide gene CDTK on human hepatocellular carcinoma cell line Bel-7402 by up-regulating the transcription activity of tumor-specific promoter h TERT. Methods: CMV enhancer, h TERT promoter and CDTK suicide fusion gene were cloned into the ribosomal gene targeting vector p Hrn to construct pT-CDTK-GFP vector and transfected into Bel-7402 cells. After RT-PCR The expression of CDTK gene and the killing effect on hepatocarcinoma cells in vitro were detected by HPLC and MTT. Then, Bel-7402 cells were inoculated into the subcutaneous tumor in nude mice and transfected into the tumor-killing vector pHr-Ce Tp CDTK-GFP for tumorigenesis. The transfected cells were inoculated with tumorigenicity and detected Its tumorigenic time and tumor growth curve, etc., from two aspects of its in vivo killing effect. Results: Tumor-specific therapeutic vector was successfully constructed. After transfected into hepatoma cells in vitro, RTPCR and HPLC confirmed the expression of CDTK gene in hepatocellular carcinoma, and the therapeutic vector was significantly toxic to hepatoma cells in vitro. The results of animal experiments found that 5-Fu serum concentration in the treatment group of tumor-bearing mice was 7.694 μg / ml, and the tumor volume in the treatment group was 6.5 times lower than that of the control group. After transfection of the vector, the tumorigenicity of tumor cells in the treatment group was 8 days later than that of the control group, and the average survival time of the untreated group was 16 days longer than that of the control group. CONCLUSION: The p-Ht-Ce Tp CDTK-GFP vector can efficiently target and kill hepatoma cells both in vitro and in vivo, providing a new approach for gene therapy of hepatocellular carcinoma.