中文摘要：

目的：用基因工程方法改造成纤维细胞，使其分泌血管内皮细胞生长因子，观察成纤维细胞作为基因转染的靶细胞的可行性。 方法：实验于2005-11在解放军第四军医大学西京医院全军整形外科研究所完成。PcDNA3．1（-）／VEGF165质粒的扩增、提取、纯化和鉴定后，将培养14d细胞融合约80％的小鼠NIH3T3细胞在24孔板中进行转染，细胞随机分为3组，每组10孔，分别为PcDNA3．1（-）／VEGF165质粒转染组．空质粒转染组和不转染质粒组。采用免疫组织化学化学检测血管内皮细胞生长因子表达。ELISA方法检测血管内皮细胞生长因子外分泌。四甲基偶氮唑盐法检测小鼠NIH3T3细胞对PcDNA3．1（-）／VEGF165质粒转染的敏感性。 结果：①PcDNA3．1（-）／VEGF165质粒的基因测序结果显示序列正确。②免疫组织化学染色显示：小鼠NIH3T3细胞转染4d后胞浆内可观察到阳性表达产物，对照组呈阴性结果。③ELISA检测结果：小鼠NIH3T3细胞转染14d后，PcDNA3．1（-）／VEGF165质粒转染组、空质粒转染组和不转染质粒组3组培养上清中血管内皮细胞生长因子浓度分别346±23，0，0ng／L（P〈0．05）。④四甲基偶氮唑盐法检测结果：PcDNA3．1（-）／VEGF165质粒转染组、空质粒转染组和不转染质粒组所测的490nm的A值比较差异均无显著性（依次为0．96±0.11，0．91±0．10，0．98±0．16，P〉0．05）。PcDNA3．1（-）／VEGF165质粒转染对小鼠NIH3T3细胞增殖无影响。 结论：小鼠NIH3T3细胞可作为血管内皮细胞生长因子基因转染的靶细胞，用于基因治疗。

英文摘要：

AIM： With gene engineering method to reconstruct fibroblast so as to make fibroblast secrete vascular endothelial cell growth factors （VEGF）. To observe the feasibility of fibroblast as target cell of gene transfection. METHODS： The experiment was performed at the Institute of Military Plastic Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA in November 2005. After amplification, extraction, purification and evaluation of plasmid PcDNA3.1 （-）/VEGF165, mouse NIH3T3 ceils cultured for 14 days and 80% conjugated was transfected in 24-well plate. The ceils were randomly assigned into 3 groups with 10 wells in each group： plasmid PcDNA3.1 （-）/VEGF165 transfection group, blank plasmid transfection group and plasmid non-transfection group. The expression of VEGF was assessed with immunohistochemical method. The external secretion of VEGF was measured with ELISA. MTT method was used to detect the effect of plasmid PcDNA3.1 （-）/VEGF165 transfection on mouse NIH3T3 ceils. RESULTS： ①Plasmid PcDNA3.1 （-）/VEGF165 sequencing result showed the sequence was correct. ②Immunohistochemical staining showed that after mouse NIH3T3 cell transfection for 4 days positive expressive products appeared in cytoplasm, and control group showed negative result. ③ELISA examination result： After mouse NIH3T3 cell transfection for 14 days, concentration of VEGF in supernatant in the plasmid PcDNA3.1（-）/ VEGF165 transfection group, blank plasmid transfection group and plasmid non-transfection group was 346±23,0 ,0ng/L, respectively （P 〈 0.05）. ④ MTT method examination： There was no significant difference of 490 nm A value in the plasmid PcDNA3.1 （-）/VEGF165 transfection group, blank plasmid transfection group and plasmid non-transfection group （0.96±0.11, 0.91 ±0.10.0.98 ±0.16.P 〉 0.05）. Plasmid PcDNA3.1 （-）/VEGF165 transfection had no effect on mouse NIH3T3 cell proliferation. CONCLUSION： Mouse NIH3T3 cell can be used as target cell for VEGF gen