节点文献
放射诱导调控腺病毒介导gfp报告基因在肿瘤细胞内的表达
The Expression of Adenoviral Mediated gfp Reporter Gene in Tumor Cells Induced and Regulated by Irradiation
【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
【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.
【Key words】 regulation of gene expression; irradiation; Erg 1 promoter; gfp reporter gene; recombinant adenovirus;
- 【文献出处】 生物化学与生物物理学报 ,Acta Biochimica Et Biophysica Sinica , 编辑部邮箱 ,2001年01期
- 【分类号】Q78
- 【被引频次】15
- 【下载频次】85