[1]张猛,邓娟,李静,等.hVEGF121基因真核表达载体的构建及其在神经干细胞中的表达[J].第三军医大学学报,2011,33(01):36-39.
 Zhang Meng,Deng Juan,Li Jing,et al.Construction of eukaryotic expression vector of human vascular endothelial growth factor for transfecting neural stem cells[J].J Third Mil Med Univ,2011,33(01):36-39.
点击复制

hVEGF121基因真核表达载体的构建及其在神经干细胞中的表达(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
33卷
期数:
2011年第01期
页码:
36-39
栏目:
论著
出版日期:
2011-01-15

文章信息/Info

Title:
Construction of eukaryotic expression vector of human vascular endothelial growth factor for transfecting neural stem cells
作者:
张猛邓娟李静王延江周华东
第三军医大学大坪医院野战外科研究所神经内科
Author(s):
Zhang MengDeng JuanLi JingWang YanjiangZhou Huadong
Department of Neurology, Institute of Surgery Research,Daping Hospital,Third Military Medical niversity,Chongqing,400042,China
关键词:
人血管内皮生长因子基因真核表达载体神经干细胞绿色荧光蛋白
Keywords:
vascular endothelial growth factor eukaryotic expression vector neural stem cells green fluorescent protein
分类号:
R322.8; R394-33; R394.2
文献标志码:
A
摘要:
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。
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 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.

参考文献/References:

张猛, 邓娟, 李静, 等. hVEGF121基因真核表达载体的构建及其在神经干细胞中的表达[J].第三军医大学学报,2011,33(1):36-39.

相似文献/References:

[1]魏勇,应大君,朱楚洪,等.人工锌指蛋白真核表达载体的构建及表达[J].第三军医大学学报,2008,30(02):134.
 WEI Yong,YING Da-jun,ZHU Chu-hong,et al.Construction of eukaryotic expressing vector of artificial zinc finger protein gene and its expression[J].J Third Mil Med Univ,2008,30(01):134.
[2]王海晶,杨和平,周向东.RNA干扰CA916798基因真核表达载体的构建与鉴定[J].第三军医大学学报,2008,30(01):23.
 WANG Hai-jing,YANG He-ping,ZHOU Xiang-dong.Construction and identification of CA916798 eukaryotic expression vector for RNA interference[J].J Third Mil Med Univ,2008,30(01):23.
[3]曾凡新,董志,傅洁民,等.人β3肾上腺素受体真核表达质粒pcDNA3.1(+)-β3-AR的构建及表达[J].第三军医大学学报,2007,29(21):2070.
 ZENG Fan-xin,DONG Zhi,FU Jie-min,et al.Construction and expression of a eukaryotic β3-adrenoceptor expression vector pcDNA3.1(+)-β3-AR[J].J Third Mil Med Univ,2007,29(01):2070.
[4]周春丽,郝进,唐书谦,等.人CTLA-4Ig融合基因减毒鼠伤寒沙门菌真核表达载体的构建及表达[J].第三军医大学学报,2006,28(03):243.
[5]田坤,钱桂生.人CD40L真核表达体系的建立[J].第三军医大学学报,2007,29(10):963.
 TIAN Kun,QIAN Gui-sheng.Construction of eukaryotic expression system of a cell surface molecule——human CD40 ligand[J].J Third Mil Med Univ,2007,29(01):963.
[6]周琪,梁后杰,阎晓初,等.RNA干扰Neuropilins-2基因真核表达载体的构建及其鉴定[J].第三军医大学学报,2007,29(08):691.
 ZHOU Qi,LIANG Hou-jie,YAN Xiao-chu,et al.Construction and effect of Neuropilins-2 eukaryotic expression vector for RNA interference[J].J Third Mil Med Univ,2007,29(01):691.
[7]滕小春,刘海峰,房殿春,等.NHE-1反义基因对胃癌细胞的酸化作用及其意义[J].第三军医大学学报,2007,29(03):240.
 TENG Xiao-chun,LIU Hai-feng,FANG Dian-chun,et al.Acidification of antisense NHE-1 gene transfection on gastric cancer cell line SGC-7901 and its significance[J].J Third Mil Med Univ,2007,29(01):240.
[8]张炜炜,王涛,艾国平,等.小鼠微小RNA miR-22真核表达载体的构建及功能初步研究[J].第三军医大学学报,2007,29(09):803.
 ZHANG Wei-wei,WANG Tao,AI Guo-ping,et al.Construction of eukaryotic expression vector of mouse microRNAs miR-22[J].J Third Mil Med Univ,2007,29(01):803.
[9]钱春荣,刘昕,刘丽莎,等.PTTG1基因上调表达对A549细胞增殖和凋亡的影响[J].第三军医大学学报,2006,28(09):961.
[10]闫国和,粟永萍,王军平,等.重组真核表达载体pEGFP-N1/PDGF-A的构建及真皮干细胞的转染[J].第三军医大学学报,2005,27(20):2005.

更新日期/Last Update: 2011-01-11