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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 study the effect of sKDR on the proliferation of human umbilical vein endothelial cells (HUVECs) in order to study the eukaryotic expression vector of sKDR gene, which can obtain soluble vascular endothelial growth factor receptor 2 (sKDR) gene. Methods The total RNA of HUVECs was extracted. The gene fragments of KDR extracellular immunoglobulin regions 1 to 3 were amplified by RT-PCR and inserted into the eukaryotic expression plasmid pcDNA3.1. The sequence of the gene was identified by liposome method The recombinant plasmid pcDNA3.1-sKDR was transfected into Lewis lung cancer cells. The expression of sKDR gene and protein was detected by RT-PCR and SDS-PAGE. The supernatant of transfected recombinant plasmid was added to the vascular endothelial growth (VEGF) -induced HUVECs medium, MTT assay was used to detect the effect of the transfected cell supernatant on the proliferation of HUVECs induced by VEGF. Results The recombinant plasmid pcDNA3.1-sKDR was successfully confirmed by restriction enzyme digestion and DNA sequencing. The recombinant plasmid was successfully transfected into Lewis lung cancer cells. The results of RT-PCR and SDS-PAGE showed that sKDR was successful in both gene and protein levels The transfection of Lewis lung carcinoma cells containing sKDR significantly inhibited the proliferation of HUVECs induced by VEGF. Conclusion The eukaryotic expression plasmid pcDNA3.1-sKDR was successfully constructed and sKDR was expressed in eukaryotic system. The expressed protein significantly inhibited the proliferation of vascular endothelial cells induced by VEGF.