PEG-PEI共聚物介导VEGF165基因转染及对内皮细胞生长的影响  

Transfection of Vascular Endothelial Growth Factor Gene VEGF165 Mediated With PEG-PEI Copolymers and Its Effect on The Growth of Endothelial Cells

作　　者：张璇[1] 潘仕荣[1] 冯敏[2] 李子俊[3] 张未[1] 罗昕[2] 

机构地区：[1]中山大学附属第一医院,广州510080 [2]中山大学药学院,广州510080 [3]广东省人民医院,广州510080

出　　处：《生物化学与生物物理进展》2007年第10期1065-1071,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30570500).~~

摘　　要：为了考察PEG-PEI共聚物作为基因载体介导VEGF165基因的能力,合成不同接枝量的PEG-PEI共聚物,考察共聚物的细胞毒性,同时采用PCR技术获得上下游含有HindⅢ和BamHⅠ酶切位点的目的基因VEGF165,与pEGFP-C1构建重组质粒pEGFP-VEGF165,将PEG-PEI作为基因载体,与pEGFP-VEGF165通过自组装成DNA复合物,使其转染脐静脉内皮细胞(HUVEc),测定发荧光细胞百分数获得转染率,利用ELISA、RT-PCR检测VEGF的表达,用MTT法考察VEGF165转染HUVEc后对内皮细胞生长的影响.结果显示,形成PEG-PEI共聚物后可显著降低PEI的细胞毒性.作为基因载体介导pEGFP-VEGF165转染HUVEc后,在荧光显微镜下可见强绿色荧光蛋白表达,转染率与接枝PEG的量及N/P有关,PEG-PEI(5-25-1)在N/P=30时转染率达到最大值,比PEI显著提高.转染后血管内皮生长因子(VEGF)蛋白表达及mRNA水平均有显著提高,且可有效地刺激内皮细胞增殖.研究表明,PEG-PEI共聚物可做为基因载体,有效地介导pEGFP-VEGF165基因的传递.In order to investigate the ability ofPEG-PEI copolymers as gene carriers for delivery ofVEGF165. A series of PEG-PEI copolymers with different PEG grafting was prepared and the cytotoxicity was evaluated. Simultaneously, the VEGF165 gene segment with Hind Ⅲ and BamH Ⅰ site was obtained by PCR, which was cloned into pEGFP-CI. PEG-PEI/pEGFP-VEGF165 complexes were formed by self-assembly and transfected HUVEc. Transfection efficiency was evaluated by measuring the percentage of cells expressing green fluorecensce protein. The VEGF expression was detected by ELISA, RT-PCR, and the effect of transfection on growth of endothelial cell was evaluated by MTT. The results suggested that the formation of PEG-PEI copolymers could help to reduce the cytotoxicity of PEI. After transfection, the strong expression of green fluorescence protein was observed by fluorescence microscopy. The transfection efficiency was influenced by the number of PEG side chains and N/P ratio. Of all copolymers tested, the transfection efficiency of PEG-PEI（5-25-1） at N/P = 30 reached a maximum, which was much higher than that of PEI. The expression of VEGF protein and mRNA increased significantly, and HUVEc proliferation was accelerated after transfection.These results indicates PEG-PEI copolymers can be used as effective gene carriers for delivery ofpEGFP-VEGF 165 gene.

关 键 词：PEG-PEI共聚物 血管内皮生长因子 脐静脉内皮细胞 基因转染 

分 类 号：Q78[生物学—分子生物学]