人血管内皮细胞生长因子基因体外转染皮肤成纤维细胞后的表达效应  

Expression of human vascular endothelial growth factor gene transfected into rabbit dermal fibroblasts in vitro

作　　者：章培标[1] 侯宜[1] 武汉[2] 周余来[1] 贾晓晶[3] 龚守良[3] 侯立中[1] 

机构地区：[1]吉林大学再生医学科学研究所 [2]吉林大学中日联谊医院骨科,吉林省长春市130032 [3]吉林大学公共卫生学院放射生物实验室,吉林省长春市130021

出　　处：《中国临床康复》2006年第13期87-89,i0005,共4页Chinese Journal of Clinical Rehabilitation

基　　金：吉林省科技厅项目(20050401-2);国家高技术研究发展计划(863计划)资助(2004AA205020);吉林大学创新基金重大项目~~

摘　　要：目的:观察外源性人血管内皮细胞生长因子基因导入正常真皮成纤维细胞后,能否表达及分泌血管内皮细胞生长因子,为进一步构建转VEGF165基因组织工程皮肤在创伤修复中的应用提供新移植物。方法:实验于2001-06/2005-06在长春吉林大学完成。①真核表达载体pcDNA3.0-hVEGF165的扩增、纯化和鉴定过程为大肠杆菌DH5a感受态细胞的制备→pcDNA3.0-hVEGF165质粒DNA细菌转化→pcDNA3.0-hVEGF165质粒大量制备(碱裂解法)→质粒纯化→质粒含量纯度测定。提取的质粒载体用EcoRⅠ和XbaⅠ双酶切,琼脂糖凝胶电泳鉴定。②选取清洁级新生日本大耳白兔2只,无菌条件下取新生兔皮肤,尽量去除皮下组织,切成宽0.3cm小条块,Ⅰ型胶原酶消化,进行真皮成纤维细胞的分离与培养。脂质体介导真核表达质粒pcDNA3.0-hVEGF165转染体外培养的兔皮肤成纤维细胞,经G418筛选,获得阳性转染细胞克隆,并进行传代扩增。③酶联免疫吸附法检测转染后不同时间段的血管内皮细胞生长因子蛋白表达水平;原位杂交显示转基因细胞内VEGF165mRNA的表达情况;流式细胞仪检测细胞周期和凋亡情况;透射电子显微镜观察转染后真皮成纤维细胞的超微结构。结果:①质粒酶切鉴定结果:提取的质粒经双酶切、琼脂糖凝胶电泳鉴定后可得到5.4kb和0.57kb两个片段,与原质粒图谱符合,表明所提取的质粒为重组质粒pcDNA3.0-VEGF165。②pcDNA3-hVEGF165基因转染真皮成纤维细胞情况:共进行15次转染试验,35孔(皿)均有G418抗性集落形成,表明pcDNA3.0-hVEGF165经脂质体介导可有效转染正常皮肤成纤维细胞。③血管内皮细胞生长因子蛋白的表达:pcDNA3.0-hVEGF165转染成纤维细胞后,24h即有较高水平的血管内皮细胞生长因子蛋白表达,高达(3280±2054)ng/L,并于48,72h呈下降趋势。④血管内皮细胞生长因子cDNA原位杂交检测结果:转染后成纤维细胞可见散在的阳性细�AIM： To observe whether the exogenous human vascular endothelial growth factor （hVEGF） can express and secrete endothelial growth factor （EGF） or not after it is guided in normal dermal fibroblasts, which is the base of gene transfected artificial skin of tissue engineering as a new substitute for wound healing. METHODS： This experiment was conducted in Jilin University from June 2001 to June 2005. ① The recombinant mammalian expression plasmid vector containing hVEGF cDNA, pcDNA3.0-hVEGF165 was amplified in DH5a, then purified, and finally identified by agarose gel electrophoresis analysis after digestion with restriction endonuclease, EcoR Ⅰ and Xba Ⅰ. ②Skin biopsies were obtained from two neonatal rabbits at the level of clean animal, chipped into small pieces （0.3 cm in width）. Dermal fibroblasts were isolated and cultured in vitro by digestion with collagenase Ⅰ. Fibroblasts were transfected with the plasmids of pcDNA3.0-VEGF165 using lipo-mediated transfection, and selected with G418 for 2 to 4 weeks. The positive clones of transfected cells were obtained and proliferated in vitro. ③The level of secreted VEGF165 protein in supernatant was determined with enzyme-linked immunosorbent assay （ELISA）; The expression of VEGF165 mRNA in transfeeted cells was analyzed with in situ hybridization; Flow eytometer（FCM） was used to detect cell cycle and apoptosis; The ultrastructure of transfeeted dermal fibroblasts was observed with transmission electron mieroseope（TEM）. RESULTS： ①Two DNA segments, about 5.4 kb and 0.57 kb in length, were obtained after digestion with EcoR Ⅰ and Xba Ⅰ . It was confirmed that the obtained vector was the recombinant plasmid pcDNA3.0-VEGF165. ②The G418-resistant colonies were pooled to generate the stable transfeeted cells in all 35 wells in 15 transfeeting tests. It indicated that the pcDNA3.0-VEGF165 could be efficiently introduced into dermal fibroblasts using lipo-mediated transfection. ③When hVEGF165 detected with ELISA in cell cu

关 键 词：生物医学工程 内皮 血管/代谢 内皮生长因子/生物合成 细胞 培养的 基因 转染 成纤维细胞 皮肤 人工 

分 类 号：R381[医药卫生—医学寄生虫学]