hVEGF_(121)基因真核表达载体的构建及其在神经干细胞中的表达

Construction of eukaryotic expression vector of human vascular endothelial growth factor for transfecting neural stem cells

目的构建真核表达载体pEGFP-N1/hVEGF121,并转染神经干细胞(neural stem cells,NSCs),以探究人血管内皮生长因子(vascular endothelial growth factor,hVEGF121)基因导入NSCs的表达变化。方法分离培养胎鼠NSCs,并行nestin、GFAP免疫染色鉴定。采用PCR法,以hVEGF121质粒为模板,扩增hVEGF121全长编码序列,克隆入载体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。倒置显微镜检测转染后的NSCs生长变化及hVEGF121在其中的表达等情况。图像分析技术计算转染效率。RT-PCR法对hVEGF121转染的NSCs进行鉴定。结果成功分离培养到NSCs,并予以鉴定。构建了hVEGF121的真核表达载体pEGFP-N1/hVEGF121,成功地将hVEGF121转染NSCs,其转染效率约50%。被转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。

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 of the transfected NSCs carrying hVEGF121 gene,neurosphere axons and axon hillocks expressed neuron marker NF-200.Conclusion hVEGF121 gene can be cloned into eukaryotic expression vector pEGFP-N1 and expressed in NSCs after transfection.