sKDR真核克隆表达及其对血管内皮细胞增殖的影响  

Construction and expression of sKDR eukaryotic expression vector and its effect on proliferation of vascular endothelial cells

作　　者：刘洪[1] 毛淑华[1,2] 邓丽聪[1] 姬玲玲[1] 徐一洲[1] 黄英[1] 

机构地区：[1]四川大学华西基础医学与法医学院,四川成都610041 [2]四川省巴中市中心医院,四川巴中636600

出　　处：《华西药学杂志》2011年第4期322-324,共3页West China Journal of Pharmaceutical Sciences

基　　金：国家自然基金项目(批准号:30570693);四川省科技厅攻关计划科研基金(2006Z09-030)

摘　　要：目的研究获取可溶性血管内皮生长因子受体2(sKDR)基因构建sKDR的真核表达载体,观察sKDR对人脐静脉内皮细胞(HUVECs)增殖的影响。方法提取HUVECs总RNA,利用RT-PCR方法扩增KDR胞外免疫球蛋白1～3区基因片段,将其插入真核表达质粒pcDNA3.1中,测序鉴定基因序列正确后,用脂质体法将重组质粒pcDNA3.1-sKDR转染Lewis肺癌细胞;采用RT-PCR和SDS-PAGE方法检测sKDR在基因和蛋白水平上的表达情况;将转染重组质粒的细胞上清液加至血管内皮生长因子(VEGF)诱导的HUVECs培养基中,采用MTT法检测转染细胞上清液对VEGF诱导的HUVECs增殖的影响。结果经酶切鉴定及基因测序证实成功获得了重组质粒pcDNA3.1-sKDR;重组质粒成功转染Lewis肺癌细胞,经RT-PCR和SDS-PAGE方法证实sKDR在基因和蛋白水平上均获得了成功表达;转染的含sKDR的Lewis肺癌细胞上清液可明显抑制VEGF诱导的HUVECs增殖。结论成功构建了真核表达质粒pcDNA3.1-sKDR,sKDR在真核系统中获得了有效表达,且表达的蛋白对VEGF诱导的血管内皮细胞增殖有明显抑制作用。OBJECTIVE To amplify soluble vascular endothelial growth factor receptor-2（sKDR） gene,construct recombined eukaryotic expression plasmid of sKDR gene and observe its effect on proliferation of human umbilicus vein endothelium cells（HUVECs）in vitro.METHODS The 1st-3 rd Ig-like domains of KDR were amplified by RT-PCR from total RNA extracted from HUVECs and then inserted into plasmid pcDNA3.1 and confirmed by sequencing.The recombinant plasmid pcDNA3.1-sKDR was transfected into Lewis lung cancer cells by Lipofectamine 2000.RT-PCR and SDS-PAGE were used to detect the expression of sKDR mRNA and protein,respectively.VEGF and culture supernatant of transfected cells were used to incubate with the HUVECs.The effect of sKDR protein on proliferation of HUVECs induced by VEGF was determined by MTT method.RESULTS The recombinant pcDNA3.1-sKDR plasmid was successfully constructed,confirmed by enzyme digestion and sequencing.Recombinant plasmid pcDNA3.1-sKDR was successfully transfected into Lewis lung cancer cells.RT-PCR and SDS-PAGE identification showed that sKDR could be expressed at the levels of gene and protein in transfected cells.Culture supernatant of transfected Lewis lung cancer cells with sKDR could significantly inhibit the proliferation of HUVECs stimulated by VEGF.CONCLUSION In this study,recombinant eukaryotic plasmid pcDNA3.1-sKDR was constructed successfully and sKDR could express in eukaryotic system.sKDR protein could remarkably inhibit the proliferation of HUVECs induced by VEGF in vitro.

关 键 词：可溶性血管内皮生长因子受体2 抗血管生成 真核表达 内皮细胞 血管内皮生长因子 

分 类 号：R96[医药卫生—药理学]