摘要
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 investigate the killing effects of adenovirus-mediated cytosine diaminase/herpes simplex virus thymidine kinase （CD/TK） on human cholangiocarcinoma cell line QBC939. Methods The aimed gene CD/TK was subcloned into shuttle vector to form the transfer plasmid pAdtrack-CMV-CD/TK, then cotransfected with adenoviral genome plasmid pAdeasy-1 into the bacterium 5183. The suitable adenovirus plasmid pAd-CD/TK was digested with Pac Ⅰ, then transfected into 293 cells to package adenovirus. The recombinant adenovirus Ad-CD/TK was purified and titred and then the infection rate and efficacy were determined in human cholangiocarcinoma cell line QBC939. Results The recombinant adenovirus Ad-CD/TK was identified with enzyme digestion and PCR, then amplified in 293 cells, purified with ultracentrifugation and titred to 1×10^11/ml. The Ad-CD-TK effectively transfected cholangiocarcinoma cell line QBC939 with 90% efficacy under multiplicity of infection （m. o. i） 100. After addition with 0.1 mmol/L 5-FC and 10 μmol/L GCV, Ad-CD/TK killed 80% of QBC939 cells under m. o. i 50 and the percentage was higher than that by using 5-FC or GCV alone. Conclusions Recombinant adenovirus Ad-CD-TK has a high infection rate and efficacy for human cholangiocarcinoma cells. The gene therapy using the adenovirus-mediated double suicide gene is a promising method for treatment of cholangiocarcinoma.
出处
《中华肝胆外科杂志》
CAS
CSCD
2006年第4期257-259,共3页
Chinese Journal of Hepatobiliary Surgery
关键词
胆管肿瘤
腺病毒载体
基因治疗
自杀基因
Bile duct neoplasms
Adenovirus vector
Gene therapy
Suicide gene
