放射诱导的一氧化氮同步放大基因电路的构建及鉴定

Construction and identification of synchronous nitric oxide-amplified gene circuits controlled by radiation

背景与目的正反馈基因电路(gene circuits)对基因表达有放大作用,目的基因在细胞内的同步化表达可进一步增强基因治疗效果。本研究旨在构建由放射诱导的一氧化氮(nitric oxide,NO)同步放大基因电路,以探索提高目的基因表达水平、延长表达时间的新途径,从而进一步完善肿瘤放射基因治疗模式。方法将具有放射诱导性的c-fos启动子与诱导性一氧化氮合酶(inducible nitric oxide synthase,i NOS)串联,构建c-fos启动子驱动的i NOS和绿色荧光蛋白(green fluorescent protein,GFP)双顺反子表达载体pfos-i NOS/GFP;用脂质体介导该载体转染肺腺癌A549细胞,予以一定剂量照射,观察照射后不同时相点细胞荧光强度,检测i NOS蛋白表达水平。结果转染载体pfos-i NOS/GFP的细胞照射后细胞荧光强度、iNOS表达水平均较对照组明显增加,其中以照射后16h增加最为明显。结论成功构建了放射诱导的NO同步放大基因电路,大大提高了放射诱导的目的基因的表达水平,为进一步提高肿瘤放射基因治疗的疗效奠定了理论基础。

Background and objective It has been proven that the positive feedback gene circuits can increase the expression level of interested genes, and the synchronization of genetic circuits can further enhance the efficacy of gene therapy. In order to obtain an enhanced and prolonged gene expression in target cells, a radiation controlled positive feedback genetic circuit is constructed via linking the c-los promoter with the inducible nitric oxide synthase （iNOS） cDNA, which can be synchronized by nitric oxide （NO） intercellular messen- ger. Ultimately, the efficacy of radiogenetic therapy for cancer will be improved. Methods Using the gene re- combination techniques, the vector pfos-iNOS/green fluorescent protein （GFP） was generated by cloning the radiation-responsive c-los promoter into the plasmid vector pIRES2-EGFP to replace the primary CMV pro- moter, and then inserting human iNOS cDNA downstream of c-los promoter in the vector pIRES2-EGFP. The constructed plasmids were then downloaded into A549 cells with lipofectamine. With exposure of various doses of ionizing radiation, outputs of GFP and iNOS in the treated cells were observed and analyzed. Results The interested plasmid was successfully constructed, proved by restriction enzyme digestion analysis. The outputs of GFP and iNOS in the transfected cells were markedly increased compared with the control cells after radiation, the peak level was seen in 16 hours after radiation. Conclusion A positive feedback genetic circuit is successfully developed, composed by e-los promoter and iNOS cDNA, which can be synchronized by secreting the intercellular messenger NO. This genetic circuit will be utilized in further study.