视网膜特异性表达人血管内皮生长因子基因载体的构建  

Construction of specifically expressed vascular endothelial growth factor_(165) gene in retina

作　　者：吕秀兰[1] 吕林[1] 李永浩[1] 张静琳[1] 李石毅[1] 

机构地区：[1]中山大学中山眼科中心,广州510060

出　　处：《中华眼底病杂志》2005年第2期106-109,共4页Chinese Journal of Ocular Fundus Diseases

基　　金：广东省重点科技攻关项目 (A0 52 0 0 1 0 84)

摘　　要：目的　构建在视网膜组织特异性表达的人血管内皮生长因子 (VEGF) 1 6 5基因。　方法　用聚合酶链反应 (PCR)方法从 BL AB/ C鼠全基因组扩增能在视网膜组织特异性表达的 rho启动子 ,经限制性内切酶纯化后克隆于质粒 pc DNA3.1 + - VEGF1 6 5中 ,建立重组质粒 pc DNA3.1 + - rho- VEGF1 6 5,通过限制性内切酶酶切分析及 PCR鉴定筛选出正确重组质粒 pc DNA3.1 + - rho- VEGF1 6 5,由 jet PEI介导转染人视网膜色素上皮细胞和人脐静脉内皮细胞 ,并通过免疫组织化学染色以及绘制细胞生长曲线检测在人视网膜色素上皮细胞和人脐静脉内皮细胞中 VEGF蛋白的表达。　结果　在人视网膜色素上皮细胞中 ,重组质粒pc DNA3.1 + - rho- VEGF1 6 5比质粒 pc DNA3.1 + - VEGF1 6 5的 VEGF蛋白表达强 ,在人脐静脉内皮细胞 ,两者的表达量无明显差别。　结论　载体 pc DNA3.1 + - rho- VEGF1 6 5的构建为进一步研究 VEGF在视网膜新生血管形成中的致病机理提供基础材料 ,并为进一步建立视网膜特异性表达 VEGF转基因鼠模型建立了基础。Objective To construct specifically expressed vascular endothelial growth factor (VEGF) 165 gene in retina. Methods Rho promoter, specifically expressed in retina, was amplified by polymerase chain reaction (PCR) from the genomic DNA of a BLAB/C rat, then it was cut with restriction enzymes and cloned into the plasmid pcDNA 3.1+-VEGF 165 to form recombinant plasmid pcDNA 3.1+-rho-VEGF 165. The correct recombinant plasmid pcDNA 3.1+-rho-VEGF 165 was identified by restriction enzymes and PCR, and was transferred by jetPEI into cultured human navel vein endothelial cells and human retinal pigment epithelial (RPE) cells. The expression of VEGF protein in human navel vein endothelial and RPE cells was detected by immunocytochemical staining and protraction of the growth curve of the cells. Results In human RPE cells, the expression of VEGF protein was more in recombinant plasmid pcDNA 3.1+-rho-VEGF 165 than that in plasmid pcDNA 3.1+-VEGF 165; in human navel vein endothelial cells, no obvious difference of the expression of VEGF protein between recombinant plasmid pcDNA 3.1+-rho-VEGF 165 and plasmid pcDNA 3.1+-VEGF 165 was found. Conclusions The construction of pcDNA 3.1+-rho-VEGF 165 carrier may provide the basic material for the study of the nosogenesis of VEGF in retinal neovascularization, and establish the foundation to set up the model of transgenic mice with VEGF specific expressing in retina.

关 键 词：VEGF165 人视网膜色素上皮细胞 PCDNA3 血管内皮生长因子 人血 人脐静脉内皮细胞 VEGF蛋白 构建 结论 目的 

分 类 号：R346[医药卫生—基础医学]