人FAS启动子驱动的HSV-TK重组腺病毒靶向杀伤SKBR3乳腺癌细胞的研究

Targeting cytotoxic effect of recombinant adenovirus carrying HSV-TK driven by FAS promoter on SKBR3 breast cancer cells

目的:构建脂肪酸合成酶(FAS)启动子驱动下的单纯疱疹病毒胸苷激酶(HSV-TK)重组腺病毒,研究其对乳腺癌细胞SKBR3的靶向杀伤作用。方法:以AdEasyTM腺病毒系统为载体,构建FAS启动子驱动下的HSV-TK重组腺病毒载体Ad-FAS-TK,将线性化的Ad-FAS-TK在AD-293细胞中包装,经过大量扩增和纯化,得到重组腺病毒。MTT法检测重组腺病毒Ad-FAS-TK与前体药物更昔洛韦(GCV)对SKBR3细胞的靶向杀伤作用。TUNEL法检测细胞凋亡。结果:成功构建FAS启动子驱动下的HSV-TK重组腺病毒载体Ad-FAS-TK,经包装、扩增和纯化得到约1010pfu/ml的重组腺病毒。MTT法和TUNEL检测结果显示,FAS启动子驱动下的HSV-TK重组腺病毒与GCV能够诱导SKBR3细胞凋亡,产生靶向细胞毒作用。结论:FAS启动子驱动下的HSV-TK重组腺病毒联合GCV对SKBR3细胞具有靶向杀伤作用,FAS启动子可以作为肿瘤靶向基因治疗的工具。

Objective：To construct the recombinant adenovirus carrying herpes simplex virus thymidine kinase （HSV -TK） driven by human fatty acid synthase （FAS） promoter and analyze its selective cytotoxic effect in vitro on human breast cancer cell line SKBR3 combined with ganciclovir （GCV）. Methods： To construct the recombinant adenovirus carrying HSV -TK driven by human FAS promoter （Ad -FAS -TK） based on AdEasyTM adenoviral system and analyze the selective cytotoxic effect of the recombinant adenovirus on SKBR3 breast cancer cells combined with prodrug GCV by MTT and TUNEL assays. Results ： We successfully constructed the adenoviral vector Ad - FAS -TK. After packing, amplification and purification, we harvested 10^10 pfu/ml recombinant adenovirus. The recombinant adenovirus combined with GCV treatment could evidently inhibit the proliferation of SKBR3 breast cancer cells and induce cell apoptosis, but showed low activity in normal fibroblast cells. Conclusion： Adenoviral - mediated suicide gene therapy controlled by FAS promoter could induce specific cytotoxic effect on SKBR3 breast cancer cells, suggesting FAS promoter can serve as a useful tool for transcriptional targeting of breast cancer gene therapy.