【摘要】 为建立放射诱导基因表达调控系统并用于肿瘤的基因治疗 ,利用PCR技术克隆出放射诱导基因Egr 1基因启动子 ,经测序证实后与报告基因gfp连接 ,并利用新型、高效的细菌内同源重组腺病毒载体制备方法制备出重组腺病毒AdEgr GFP。感染腺病毒的肿瘤细胞给予不同剂量的γ射线照射 ,体外采用FACS方法检测GFP的表达发现 ,照射可明显提高GFP表达 ,并呈剂量依赖性 ,Western印迹检测也显示类似的结果。为进行体内实验 ,瘤内注射AdEgr GFP腺病毒后 48h ,肿瘤局部接受不同剂量的γ射线照射 ,8h后制备肿瘤组织标本用于分析GFP的表达。肿瘤组织图像分析结果显示 ,γ射线照射可显著提高肿瘤组织中GFP的表达 ,并呈剂量依赖性。结果说明 ,放射经Egr 1启动子可有效调控腺病毒介导 gfp基因的肿瘤细胞内表达更多还原

【Abstract】 In order to establish a radiation inducible gene expression system for cancer gene therapy, the promoter sequence of radiation inducible Egr 1 gene was amplified from genomic DNA of BALB/c mouse with PCR method, and linked to gfp reporter gene. Then the p Egr gfp expression cassette was subcloned into an adenoviral shuttle plasmid to generate recombinant adenovirus of AdEgr GFP by using a novel, high efficient method of homologous recombination in bacteria. After infection with AdEgr GFP, MM45T.Li tumor cells were exposed to different doses of γ irradiation from 0 Gy to 15 Gy in vitro . The percentage of GFP expression positive cells increased greatly in a dose dependent manner as detected by FACS and Western blot analysis. For in vivo study, AdEgr GFP were injected intratumorally, and tumor site received different doses of local γ irradiation 48 h after injection, and after 8 h the tumor samples were biopsed for investigating the GFP expression. Tumor tissue image analysis revealed that γ irradiation could markedly increase GFP expression in a dose dependent manner as compared with that of non irradiated control group. Our results indicate that the irradiation can effectively control adenoviral mediated GFP expression in tumor cells via Egr 1 promoter, and these data laid basis for further gene radiotherapy study.更多还原