重组腺病毒Ad-CD/TK对人肝癌细胞的作用

Effect of recombinant adenoviruses with CD/TK fusion suicide gene on human hepatocellular carcinoma cells

目的 :通过构建含CD/TK双自杀基因的重组腺病毒 ,探讨治疗肝细胞癌的途径。方法 :目的基因CD/TK ,自真核载体 pCEA CD/TK中切出 ,亚克隆至腺病毒穿梭质粒中 ,构建成转移质粒pAdtrack CMV CD/TK ,经PmeⅠ酶切后与 pAdeasy 1质粒混匀共转化细菌BJ5 183,挑选克隆pAd CD/TK ,经PacⅠ酶切后转染 2 93细胞 ,PCR鉴定后 ,在人肝癌细胞中测定其感染效率并观察其体外对人肝癌细胞的作用。结果 :pAdtrack CMV CD/TK和 pAd CD/TK经酶切鉴定正确 ,pAd CD/TK转染2 93细胞 ,制备腺病毒后 ,发现其对人肝癌细胞有较高的感染效率 ,给予氟胞嘧啶后 ,发现被感染细胞能够被杀死 ,并具有很高的旁观者效应。结论 :构建的含CD/TK双自杀基因的重组腺病毒对人肝癌细胞有较高的感染效率 。

Objective:To investigate gene-therapy for human hepatocellular carcinoma with adenovirus vectors by double suicide gene CD/TK. Methods:Double suicide gene CD/TK was liberated from eukaryotic vectors pCEA-CD/TK and subcloned into shuttle vectors, and the transfer plasmid pAdtrack-CMV-CD/TK was formed after linearizing with Pac 1. It was recombinated with pAdeasy-1 in bacteria BJ5183. The identified adenovirus plasmid was digested by Pac1 and was transfected into 293 cells to pack the adenoviruses. After PCR determination, its titre was measured, and the infection rate and efficacy were tested in human hepatocellular carcinoma cells. Results:pAdtrack-CMV-CD/TK and pAd-CD/TK were tested by endonuclease digestion. Ad-CD/TK was produced in 293 cells, and the human hepatocellular carcinoma cells (SMMC7721) infected by Ad-CD/TK were killed after 5-FC was used, and bystander effects were observed. Conclusion:Recombinant adenoviruses with CD/TK fusion suicide gene have a high infection rate and efficacy for human hepatocellular carcinoma cells.