放射诱导HSV-TK基因在肺癌细胞中靶向、高效性表达的研究

Lung carcinoma targeted expression of HSV-TK gene controlled by radioinducible synchronizing positive feedback genetic circuit

目的构建放射诱导的同步放大基因电路,通过基因电路调控HSV-TK基因在肺癌细胞中靶向、高效性表达,以提高放射-基因治疗的有效性和靶向性。方法利用分子克隆的方法将HSV-TK基因插入到同步放大基因电路载体的下游,并采用脂质体介导重组载体和对照载体转染肺癌细胞A549;G418筛选阳性克隆,转染细胞给予2Gy X线照射后,检测照射前后不同时相点细胞内TK表达情况,及不同浓度前药GCV对转染重组载体和对照载体的肺癌细胞的生长抑制率。结果照射后转染同步放大重组载体的细胞内TK的表达明显强于对照组,且随时间的推移而逐渐增强,以照射后16h最为明显。同一浓度的GCV对转染同步放大载体的A549细胞的抑制率明显高于对照组和未转染组,IC50也明显低于对照组和未转染组。结论同步放大基因电路明显增强了靶细胞内HSV-TK基因的表达,进一步完善了放射-基因治疗这一新的肿瘤治疗模式。

Objective Previous studies have shown that the radioinducible synchronizing positive feedback genetic circuit via linking the c-fos promoter with the iNOS（inducible nitric oxide synthase） cDNA 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 circuirt 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 vitro. Methods Using the gene recombination techniques, the fragment of TK gene was cloned into the downstream of vector pfos-iNOS/GFP, and then an cancer cell line A549 was transfected with the recombinant vector and controlled vector. Following selection by G418 medium , and resistant clones were identified by RT-PCR . The exposure of ionizing radiation and different concentration of GCV was given to the transfected cells and untransfected A549 cells. The killing effect of HSV-TK/GCV was observed and analyzed by MTT test. Results Expression of HSV-TK gene in A549 cells infected with pfos-iNOS/TK was markedly increased after irradiation in a timedependent manner,with a peak at 16h post irradiation. In vitro gene therapy experiment,higher sensitivity to GCV was observed in A549 cell transfected with pfos-iNOS/TK after irradiation,but not in controlled cells and untransfected cells. Conclusion Tumor targeted expression of HSV-TK gene under control of radioinducible synchronizing positive feedback genetic circuit, represents a novel strategy for safe and effective radio-genetic therapy of lung carcinoma, and might bring more hope for the patients with carcinoma.