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;毛淑华;邓丽聪;姬玲玲;徐一洲;黄英;
Author：
收稿日期： 年卷（期）页码：2011,26(04):-322-324
期刊名称：华西药学杂志
Journal Name：WEST CHINA JOURNAL OF PHARMACEUTICAL SCIENCES
关键字：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
Key words：
基金项目：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)
中文摘要
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。
参考文献
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