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目的观察腺病毒介导的胞嘧啶脱氨酶/单纯疱疹病毒-1-胸苷激酶(CD/TK)双自杀基因对人胆管癌细胞 QBC939的体外杀伤作用。方法 CD/TK 克隆入穿梭载体成为 pAdtrack-CMV-CD/TK,与骨架载体 pAdeasy-1在细菌内同源重组为 pAd-CD/TK,经 PacⅠ酶切,293细胞包装、扩增、纯化后,体外转染人胆管癌细胞 QBC939,并给予前药5-FC 或 GCV,观察其体外杀伤效果。结果含 CD/TK 基因的重组腺病毒鉴定正确,扩增纯化后,病毒滴度为1×10~(11)颗粒/ml。重组腺病毒对 QBC939细胞在感染倍数(m.o.i)为100时的转染效率为90%,在 m.o.i50感染时,0.1mmool/L的5-FC 及10 μmol/L 的 GCV 对 QBC939细胞的杀伤率为80%,明显高于单用5-FC 与 GCV 的效应。结论双自杀基因以腺病毒为载体对人胆管癌细胞转染效率高,体外杀伤效应明显。腺病毒介导的双自杀基因治疗有望成为治疗胆管癌的有效方法。
Objective To observe the in vitro cytotoxicity of adenovirus mediated cytosine deaminase / herpes simplex virus-1-thymidine kinase (CD / TK) double suicide gene on human cholangiocarcinoma cell line QBC939. Methods The shuttle vector pAdtrack-CMV-CD / TK was cloned into pAdtrack-CMV-CD / TK vector and homologously recombined into pAddeasy-1 vector to construct pAd-CD / TK vector. After PacI restriction endonuclease digestion, 293 cells were packaged and amplified. , In vitro transfected human cholangiocarcinoma cells QBC939, and given prodrug 5-FC or GCV to observe its in vitro killing effect. Results The recombinant adenovirus containing CD / TK gene was identified correctly. After amplification and purification, the virus titer was 1 × 10-11 particles / ml. The transfection efficiency of QBC939 cells was 100% at the fold of 100 (moi) of recombinant adenovirus was 90%. The killing rate of QBC939 cells by 0.1mmool / L 5-FC and 10μmol / L GCV at moi50 infection 80%, significantly higher than the effect of single-use 5-FC and GCV. Conclusion Double suicide gene with adenovirus vector transfected human cholangiocarcinoma cells with high efficiency, in vitro killing effect is obvious. Adenovirus-mediated double suicide gene therapy is expected to be an effective method for the treatment of cholangiocarcinoma.