放射控导正反馈基因环路对肺癌wt-p53基因靶向性表达的研究  

lung cancer targeted expression of wt-p53 gene controlled by positive feedback gene circuits which regulated by irradiation

作　　者：毛丽伟[1] 王卫东[1] 李德志[1] 陈正堂[1] 

机构地区：[1]第三军医大学新桥医院全军肿瘤研究所,重庆400037

出　　处：《重庆医学》2007年第19期1921-1923,共3页Chongqing medicine

基　　金：国家自然科学基金(30300097)。

摘　　要：目的构建pE_6R_4/p53/GFP重组质粒并检测其在人非小细胞肺癌H1299(p53—/—)细胞的辐射诱导表达。方法用嵌合型增强子E_6R_4取代pci-neo质粒的CMV增强子,将wt-p53cDNA全长序列、IRES_2-EGFP报告基因片段构建于嵌合型增强子E_6R_4下游,获得pE_6R_4/p53/GFP重组质粒,采用脂质体法将重组质粒转染人非小细胞肺癌H1299(p53—/—)细胞;采用RT-PCR方法鉴定wt-p53基因整合情况;Western-blot方法检测不同辐射剂量8mVX线照射后,被转染细胞中wt-p53蛋白表达水平和持续时间。结果酶切和测序鉴定pE_6R_4/p53/GFP重组质粒构建正确;不同剂量8mVX线照射后,4Gy照射剂量时wt- p53基因表达最强,且持续时间延长。结论成功将辐射反应元件和正反馈基因环路技术相结合,实现了wt-p53基因的表达调控,为进一步研究非小细胞肺癌的放射-基因联合治疗奠定了基础。Objective To construct a recombinat plasmid pE6R4/p53/GFP and investigate the expression of wt-p53 gene induced in non-small lung cancer H1299（p53-/-） cells. Methods The CMV enhancer of pci-neo plasmid were replaced by mosaicism enhancer E6 R4. Wt-p53 cDNA and IRES2-EGFP reported gene was ligated to the downstream of enhancer E6R4 to constructed pE6R4/p53/GFP plasmid, The recombinant plasmid transfected into H1299 （p53-/-） cells with lipofectamine 2000. Analyzing gene integration with RT PCR. Western blot method was used to detect the level of wt-p53 protein expression and persistence time after different 8MV X-ray irradiation dose. Results Restrictive enzyme digestion and sequencing showed the recombinant pE6R4/ p53/GFP was reconstructed correctly. After different irradiation dose, the expression of wt-p53 gene was the highest after 4Gy irra diation dose and the expression time was prolonged. Conclusion Radiation response element was combined with positive feedback gene circuits successfully to control the expression of wt-p53 gene. It could provide experimental data in the field of non-small lung cancer radiation-gene therapy.

关 键 词：基因环路 X线 非小细胞肺癌细胞 

分 类 号：R734.2[医药卫生—肿瘤] R730.21[医药卫生—临床医学]