人血管生成素-1基因真核表达载体的构建及其在人脐带间充质干细胞中的表达  

Construction of human angiopoietin-1 fukaryotic expression vecter and its expression in human umbilical mesenchymal stem cells

作　　者：萧鸿[1] 刘帅[2] 李群秀[2] 张克剑[2] 侯阳[2] 宋慧娟 刘学政[2] 

机构地区：[1]辽宁医学院附属第一医院教务科,锦州121001 [2]辽宁医学院人体解剖学教研室,锦州121001 [3]高校分子细胞生物学与新药开发重点实验室

出　　处：《眼科研究》2010年第9期846-850,共5页Chinese Ophthalmic Research

基　　金：辽宁省科学技术计划项目(2003225007)

摘　　要：目的构建人血管生成素-1(Ang-1)基因的真核表达载体,并检测其在人脐带间充质干细胞(UC-MSCs)中的表达,为进一步研究Ang-1防治糖尿病视网膜病变(DR)的血管渗漏提供实验依据。方法提取人胎盘组织中的总RNA,用逆转录-聚合酶链反应(RT-PCR)特异性扩增Ang-1编码区序列,亚克隆到真核表达载体pEGFP-N1。将pEGFP-N1-Ang-1导入UC-MSCs,然后应用含质量分数10%胎牛血清的DMEM培养基对人UC-MSCs进行培养,荧光显微镜下观察Ang-1在人UC-MSCs中的表达,Western blot法检测重组体的表达。结果 RT-PCR法成功克隆人Ang-1基因编码区序列,纯度为1.82;PCR扩增的片段长度为1.5 kb,与预期片段大小相近。双酶切鉴定法可识别重组pEGFP-N1-Ang-1转染质粒,目的基因与预期基因序列的同源性为100%。人UC-MSCs转染的pEGFP-N1/Ang-1质粒能够表达Ang-1蛋白,呈现绿色荧光。转染pEGFP-N1的人UC-MSCs和对照组人UC-MSCs均未见相应的Ang-1蛋白表达。结论成功地克隆出人的Ang-1基因并构建了真核表达载体pEGFP-N1-Ang-1,为将Ang-1基因转染至人UC-MSCs治疗DR奠定了基础。Background Angiopoietins（Angs） can be calssified into Ang-1,Ang-2,Ang-3 and Ang-4 based on their different sequence of amino acid.Ang-1/Tie system can inhibite neovascularization and decrease the leakage of retinal vessel in diabetic retinopathy（DR） and is a deal intervene site.ObjectivePurpose of this study was to construct the recombinant eukaryotic expression vector pEGFP-N1-Ang-1 and investigate its transient expression in human umbilical mesenchymal stem cells（UC-MSCs）for discussing the mechanism of inhibiting the leakage of blood vessel in DR.MethodsTotal RNA was extracted from 100mg human placenta by TRIZOL reagents.The Ang-1 cDNA fragment was amplified by RT-PCR and the DNA fragment was subcloned into pEGFP-N1 ang sequenced analysis was carried out.Then,pEGFP-N1-Ang-1 was transfected into the human UC-MSCs.Human UC-MSCs were cultured in DMEM containing 10% fetal bovine serum.Expression of recombinant plasmid of pEGFP-N1-Ang-1 in human UC-MSCs was detected by Western blotting under the fluorescence microscope.This study followed the Declaration of Helsinki and was approved by the Ethic Committee of Liaoning Medical School.Written informed consent was obtained at the initial of medical procedure.ResultsThe full-length sequence of human Ang-1 gene was obtained from human placenta with the good purity（A260/A280=1.82）.The CDS of human Ang-1 gene was cloned with the specific 1.5 kb of amplified fragment and expectant fragment.On the framework of pEGFP-N1 expression vector,recombinant plasmid of pEGFP-N1-Ang-1 was identified by double-enzyme cleavage method.The sequencing result showed a 100% homology between target gene and well-known Ang-1 gene.Cultured human UC-MSCs showed the consistent shape with MSCs derived from marrow and hematoma of cord.human UC-MSCs transfected pEGFP-N1/Ang-1 plasmid could express Ang-1protein,presenting the green fluorescence.However,no Ang-1protein was expressed in human UC-MSCs transfected pEGFP-N1 and control human UC-MSCs.ConclusionEukaryotic plasmid pEGFP-N1-Ang-

关 键 词：人血管生成素-1 PEGFP-N1 pEGFP-N1-Ang-1 人脐带间充质干细胞 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]