PDGF-A链基因的克隆重组真核表达载体-第三军医大学学报.DOCVIP

hVEGF121基因真核表达载体的构建及其在神经干细胞中的表达 张 猛，邓 娟，李 静，王延江，周华东 （400042 重庆，第三军医大学大坪医院野战外科研究所神经内科） [摘要] 目的 构建真核表达载体pEGFP-N1(neural stem cells，NSCs)，以探究ascular endothelial growth factor，hVEGF121)基因导入NSCs的表达pMD18-T，随后又将其亚克隆入pEGFP-N1载体中,构建其真核表达载体pEGFP-N1/hVEGF121，并以Fugene 6介导转染，将hVEGF121导入NSCs。倒置显微镜转染后的NSCs生长变化及在其中的表达等情况。图像计算转染效率RT-PCR法对转染的NSCs进行鉴定。pEGFP-N1/hVEGF121，成功地将hVEGF121转染NSCs其转染效率约5%。NSCs生长良好。转染hVEGF121基因的NSCs被诱导分化后，神经球的轴突和轴丘表达神经元标志物NF-200。结论 成功地将hVEGF121克隆到pEGFP-N1载体中，并实现了hVEGF121基因在NSCs的表达。 [关键词] 人血管内皮生长因子基因Construction of eukaryotic expression vector of human vascular endothelial growth factor for transfecting neural stem cells Zhang Meng，Deng Juan，Li Jing，Wang Yanjiang，Zhou Huadong(Department of Neurology, Institute of Surgery Research,Daping Hospital,Third Military Medical University,Chongqing,400042,China) [Abstract] Objective To construct eukaryotic expression vector pEGFP-N1/hVEGF121 for transfecting neural stem cells (NSCs), and to investigate the expression of human vascular endothelial growth factor (hVEGF121) in transfected NSCs. Methods NSCs were isolated from fetal mice and cultured, and then identified by immunohistochemical staining of nestin and GFAP. Full-length hVEGF121 cDNA was amplified by PCR using hVEGF121 plasmid as template. The amplified cDNA fragments were cloned into plasmid vector pMD18-T, and then subcloned into eukaryotic expression vector pEGFP-N1 to construct pEGFP-N1/hVEGF121, which was identified by sequencing. Plasmid pEGFP-N1/hVEGF121 was used to transfect NSCs with the help of Fugene 6 reagent. The growth of transfected NSCs and the expression of enhanced green fluorescent protein (EGFP) were observed. Image analysis was employed to calculate the transfection efficiency. The expression of hVEGF121 in transfected NSCs was identified by RT-PCR. Results NSCs were isolated and identified. The transfection efficiency of NSCs by pEGFP-N1/hVEGF121 was approximately 50%. The transfected NSCs grew well. After the induced differentiation o