pcDNA3.1-VEGF165质粒转染骨髓基质干细胞后血管内皮生长因子的表达

Expression of vascular endothelial growth factor after transfection of pcDNA3.1-VEGF165 plasmid into mesenchymal stem cells

目的:检测pcDNA3.1-VEGF165载体转染对兔骨髓基质干细胞血管内皮生长因子表达的影响。方法:实验于2005-07/2006-01在山东省青岛市市立医院中心试验室完成。①pcDNA3.1-VEGF165质粒由华中科技大学同济医学院附属协和医院骨科杨述华教授惠赠。②选取1月龄新西兰大白兔1只,无菌条件下取胫骨和股骨,进行骨髓基质干细胞的分离与培养。③转染前24h计数细胞,每孔5×105细胞接种于6孔板,待骨髓基质干细胞达到80%～90%融合时准备转染,设立未转染组、空载体组和载体组。未转染组无特殊处理,空载体组转染pcDNA3.1载体,载体组转染pcDNA3.1-VEGF165。④转染后72h,反转录聚合酶链反应检测各组骨髓基质干细胞中血管内皮生长因子165mRNA的表达;酶联免疫吸附法检测各组骨髓基质干细胞中血管内皮生长因子165蛋白的含量。结果:①骨髓基质细胞分离培养形态观察:初始分离的骨髓细胞呈圆形,大小不一;24h后有少量细胞贴壁;48h后贴壁细胞部分为成纤维样细胞;72h贴壁的成纤维样细胞数不断增加;96h后贴壁生长的细胞主要为梭形的成纤维样细胞;分瓶后细胞形成克隆;传代后细胞贴壁生长,分裂相增多,并不断增殖分化形成均一的梭形细胞。②转染72h后各组细胞血管内皮生长因子165mRNA的表达情况:反转录聚合酶链反应检测到载体组在576bp处有明显条带,空载体组和未转染组均未见血管内皮生长因子165表达。③转染72h后各组细胞血管内皮生长因子165蛋白含量检测结果:酶联免疫吸附结果显示,载体组转染pcDNA3.1-VEGF165载体的骨髓基质细胞培养上清液中血管内皮生长因子165蛋白浓度为(170.1±14.3)ng/L,而空载体组与未转染组均未检测到血管内皮生长因子165蛋白表达,差异有显著性意义(t均=42.206,P=0.000)。结论:应用pcDNA3.1-VEGF165载体转染,可使兔骨髓基质干细胞获得外源性血管内皮生长因子165基因和蛋白的表达。

AIM： To detect the expression of VEGF mRNA and protein in rabbit mesenchymal stem cells （MSCs） transferred with pcDNA3.1-VEGF165. METHODS： The experiment was conducted in the Central Laboratory of Qingdao Municipal Hospital frorn July 2005 to January 2006. （1） The pcDNA3.1-VEGF165 plasmid was given by Professor Yang Shu-hua （Department of Orthopedics, Xiehe Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology）. （2）MSCs aseptically obtained from the femur and tibia of a New Zealand rabbit were cultured and separated in vitro. （3） Cells were numbered 24 hours before transfection, and cells were inoculated into the 6-mesh board with each pore 5×10^5 cells, pcDNA3.1- VEGF165 was transfected into the mesenchymal stem cells by liposome mediated at the time of 80%-90% confluence, and the MSCs were divided into three groups： the non-transfection group, the blank plasmid group and plasmid group. The cells in the non-transfection group received no special treatment, while cells in the blank plasmid group were transfected with pcDNA3.1 and cells in the plasmid group were transfected with pcDNA3.1-VEGF165. （4） At 72 hours after transfection, the expressions of mRNA and protein of VEGF165 gene in MSCs were analyzed by RT-PCR and ELLISA methods in three groups. RESULTS： （1） Observation on the shape of MSCs： MSCs were round initially after isolation and the volumes of the cells were different; 24 hours later, there were some cells adherent to the wall; 48 hours later, part of the adherent cells became fibroblasts; 72 hours later, the number of the adherent fibroblasts increased continuously; 96 hours later, most of the adherent cells were fusiform fibroblasts. After replanted the cell clones were formed. These clones were detached and passaged into flasks, then the cells adhered to the flasks and differentiated into homegeneous fusiform fibroblasts. （2） The expressions of VEGF165 mRNA in each group at 72 hours after transfection by RT-PCR： There was obvious mark at 576 bp in transfection group, but there was no expression of VEGF165 in plasmid group and blank plasmid group. （3） The expression of VEGF165 protein in each group at 72 hours after transfection by ELLISA： The content of VEGF in the plasmid group was （170.1±14.3） ng/L, and there was no expression of VEGF in the non-transfection group nor in the blank plasmid group, and the differences were significant（tall =42.206, P= 0.000）. CONCLUSION： pcDNA3.1-VEGF165 vector transfection can obtain the expression of exogenous VEGF165 mRNA and protein in rabbit MSCs.