人血管内皮生长因子165基因慢病毒载体的构建及其在人胚胎神经干细胞中的表达  

Construction of lentiviral vector encoding human vascular endothelial growth factor165 gene and its expression in human neural stem cells

作　　者：敬华军[1] 张宇[1] 王晓斌[1] 

机构地区：[1]温州医学院附属第一医院神经外科,325000

出　　处：《实用医学杂志》2013年第5期694-696,共3页The Journal of Practical Medicine

基　　金：浙江省自然科学基金项目(编号:Y2110807)

摘　　要：目的:构建携带人血管内皮生长因子165(hVEGF165)基因的慢病毒表达载体,转染人胚胎神经干细胞(hNSCs)并检测hVEGF165基因在hNSCs内的表达。方法:用RT-PCR法扩增出hVEGF165 cDNA,插入pCDH-CMV-copGFP慢病毒载体中,构建pCDH-hVEGF165慢病毒表达载体,双酶切和测序鉴定,包装并转染hNSCs。荧光显微镜观察转染细胞绿色荧光蛋白(GFP)表达,流式细胞术(FCM)测定转染率,Western blot法检测细胞中hVEGF165的表达。结果:酶切及测序结果显示成功构建pCDH-hVEGF165重组慢病毒表达载体,转染7d后观察宿主细胞内有大量GFP表达,FCM测定转染率为63%,Western blot检测证实hVEGF165蛋白在hNSCs中过表达。结论:本实验成功构建了携带hVEGF165的慢病毒表达载体,并在hNSCs中过表达hVEGF165基因。Objective To construct the lentivirus vectors carrying human vascular endothelial growth factor165 （hVEGF165） genes, and investigate their expression in human neural stem cells （hNSCs）. Methods hVEGF165 genes were acquired by RT-PCR,and then subcloned into the expression plasmid of pCDH-CMV- copGFP to generate the recombinant lentiviral vector pCDH-hVEGF165. The lentiviral vector pCDH-hVEGF165 was identified by restriction enzyme digestion and sequencing analysis. The recombinant lentiviral vector was used to pack and infect hNSCs. Green fluorescent protein （GFP） expression in infected hNSCs was observed under fluorescence microscope and transfection efficiency was tested by flow cytometry （FCM）. hVEGF165 expression in hNSCs was detected by Western blot analysis. Results hVEGF165 gene fragment was constructed into the pCDH- CMV-copGFP vector successfully. Enzyme digestion got correct length of hVEGF165 gene; DNA sequencing analysis confirmed that hVEGF165 gene sequence was exactly the same with that reported in GenBank. pCDH- hVEGF165 infected cells could show green fluorescence under fluorescence microscope. The transfection efficiency is 63% according to FCM. Western blot analysis confirmed hVEGF165 expression in pCDH-hVEGF165 infected hNSCs. Conclusion We successfully constructed the lentivirus vectors carrying hVEGF165 gene and obtained the hVEGF165 overexpression hNSCs.

关 键 词：血管内皮生长因子类 慢病毒载体 神经干细胞 转染 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]