pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒的构建及其在体外人脐静脉内皮细胞中抑制Ang2/Tie2基因的表达  

Construction of pSilencer 1.0-U6-Ang2/Tie2-siRNA expressing plasmid vectors and the inhibitory effects on the expression of Ang2/Tie2 in HUVECs

作　　者：王彪[1] 郭国祥[1] 林菊丽[1] 单秀英[1] 张声[2] 鲁开化[3] 

机构地区：[1]福建医科大学附属第一医院整形外科,福建福州350005 [2]福建医科大学附属第一医院病理科,福建福州350005 [3]第四军医大学西京医院整形外科研究所,陕西西安710043

出　　处：《中国现代医学杂志》2011年第5期590-595,603,共7页China Journal of Modern Medicine

基　　金：卫生部科学研究基金-福建省卫生教育联合攻关计划(WKJ2008-2-51);福建省自然科学基金计划项目(No:2007J0269);福建省教育厅科技项目(No:JB08088)

摘　　要：目的探讨应用RNA干扰(RNAi)技术抑制Ang2、Tie2基因的表达情况,为进一步研究其在体外抑制血管生成以及抑制肿瘤血管生成的动物实验研究奠定基础。方法构建pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒各2条并予以鉴定。用pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒转染人脐静脉内皮细胞(HUVECs),采用免疫细胞化学染色和RT-PCR方法检测各组HUVECs中Ang2及Tie2的蛋白及mRNA的表达状况。结果成功构建pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒各2条,用其转染HUVECs后,免疫细胞化学染色和RT-PCR检测结果显示,Ang2、Tie2在蛋白和mRNA的表达水平均受到明显抑制(P<0.05),并且siRNA的2条重组质粒之间均差异无统计学意义(P>0.05)。结论 pSilencer1.0-U6-Ang2/Tie2-siRNA在体外能够抑制HUVECs的Ang2和Tie2的蛋白及mRNA的表达。【Objective】 To investigate the inhibitory effects on the expression of Ang2/Tie2 in HUVECS using the RNA interference technologies.The result offered in vitro evidence for further in vivo animal studies to inhibit angiogenesis by RNA interference.【Methods】 Two plasmids,pSilencer1.0-U6-Ang2-siRNA-1,2 and pSilencer 1.0-U6-Tie2-siRNA-1,2 were constructed and identified.The protein and mRNA expression levels of Ang2/Tie2 in-HUVECs transfected with these plasmids were investigated by immunohistochemistry and RT-PCR.【Results】 The recombinant plasmids pSilencer1.0-U6-Ang2/Tie2-siRNA were constructed successfully,and the immunohistochemistry and RT-PCR showed that Ang2/Tie2 expression in the transfected cells were down-regulated significantly for the recombinant plasmids at both protein and mRNA levels（P 0.05）.The expression levels were not significantly different（P 0.05） between the two plasmids of each gene.【Conclusion】 The plasmids of pSilencer1.0-U6-Ang2/Tie2-siRNA could inhibit Ang2/Tie2 expression in HUVECs cultured in vitro.

关 键 词：RNA干扰 促血管生成素2 受体Tie2 质粒载体 脐静脉内皮细胞 

分 类 号：Q782[生物学—分子生物学] Q786