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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 a human vascular endothelial growth factor (VEGF) 165 gene that is specifically expressed in the retina. Methods The rho promoter specific for retinal tissue was amplified from whole genome of BL AB / C mice by polymerase chain reaction (PCR) and purified by restriction endonuclease. The rho promoter was cloned into pcDNA3.1 + - VEGF1 6 5, the recombinant plasmid pcDNA3.1 + - rho-VEGF165 was established and the correct recombinant plasmid pcDNA3.1 + - rho-VEGF1 65 was screened by restriction enzyme digestion and PCR. PEI-mediated transfection of human retinal pigment epithelial cells and human umbilical vein endothelial cells, and immunohistochemical staining and cell growth curve detection of human retinal pigment epithelial cells and human umbilical vein endothelial cells VEGF protein expression. Results In human retinal pigment epithelial cells, the expression of VEGF protein of recombinant plasmid pcDNA3.1 + - rho - VEGF165 was stronger than that of pcDNA3.1 + - VEGF165 in human umbilical vein endothelial cells No significant difference in volume. Conclusion The construction of pcDNA3.1 + - rho - VEGF165 will provide the basic information for further study of the pathogenesis of retinal neovascularization and provide a basis for further establishing a retinal - specific transgenic mouse model of VEGF.