放射诱导同步放大基因电路调控HSV-TK表达对肺癌移植瘤的抑制作用

Effects of radioinducible synchronizing positive feedback genetic circuit on A549 subcutaneous xenograft in nude mice

目的构建由放射诱导的一氧化氮(NO)同步放大基因电路,并利用该基因电路调控单纯疱疹病毒胸苷激酶(HSV-TK)表达,以研究该基因电路对肺癌细胞体内的治疗作用。方法利用分子克隆的方法构建重组治疗载体pfos-iNOS/TK和对照载体pfos-TK/GFP,并采用脂质体介导重组治疗载体和对照载体转染肺癌细胞A549;G418筛选阳性克隆;建立BALB/c裸鼠移植瘤模型,检测接受照射和未接受照射的瘤组织内TK表达情况,分析给予照射和更昔洛韦(GCV)后肿瘤体积的变化。结果接受照射的瘤组织内TK表达明显强于未接受照射组,接受照射的转染治疗载体组强于接受照射的转染对照载体组,放射合并GCV可明显抑制转染治疗载体的移植瘤,且抑瘤率明显高于单纯照射、GCV治疗和转染对照载体的对照组。结论放射诱导的同步放大基因电路可明显提高体内转染细胞TK的表达,对肺癌移植瘤有显著的抑制作用,明显地提高了HSV-TK/GCV系统对肺癌细胞的敏感性,进一步完善了肿瘤的放射—基因治疗模式。

Objective Previous studies have shown that the radioinducible synchronizing positive feedback genetic circuit via linking the c-los promoter with the iNOS （ Inducible nitric oxide synthase） eDNA can increase the expression level of interested genes significantly. Then the genetic circuit was linked with HSV-TK （herpes simplex virus type Ⅰ thymidine kinase） in order to further study the regulation of the genetic circuit on the expression and killing effect of a suicide gene therapy system of HSV-TK/GCV （herpes simplex virus type Ⅰ thymidine kinase/ganciclovir） for lung cancer cell A549 in vivo. Methods Using tie gene recombination techniques, the fragment of TK gene was cloned into the downstream of vectors pfos-iNOS/GFP and multiple cloning sites of vector pfos-GFP to form the therapeutic vector pfos-iNOS/TK and control vector pfos-TK/GFP, and then a cancer cell line A549 was transfected with the therapeutic vector and control vector. Following selection by G418 medium, expression of TK in tumor tissue formed by transfected cells invo was analyzed by western blot. Results Level of exprexsion of TK in transfected cells in vivo was raised after irradiation. Level of expression of TK was higher in A549 cells transfected with therapeutic vector in vivo than that in cells transfected with control vector after irradiation. Growti of tumor tissue formed by cells transfected with therapeutic vector was obviously inhibited as given GCV and irradiation. Rate of tumor inhibination in given GCV and irradiation group to cells transfected with therapeutic vector in vivo was much higher than that in only given GCV group,only irradiation group and control group. Conclusion Radioinducible synchronizing positive feedback genetic circuit can improve expression of TK, sensitivity of HSV-TK/GCV to lung carcinoma cells and rate of tumor inhibination in vivo and repre- sents a novel strategy for more effective radio-genetic therapy of lung carcinoma.