可溶性血管内皮生长因子受体1真核克隆表达及其对血管内皮细胞增殖的影响  

Construction and Expression of Soluble Vascular Endothelial Growth Factor Receptor-1 Eukaryotic Expression Vector and Its Effect on Proliferation of Vascular Endothelial Cells

作　　者：姬玲玲[1] 毛淑华[1] 刘洪[1,2] 许士叶[1] 杨雨[3] 易成[3] 黄英[1] 

机构地区：[1]四川大学华西基础医学与法医学院病理生理学教研室,成都610041 [2]成都医学院门诊部,成都610083 [3]四川大学华西医院腹部肿瘤科,成都610041

出　　处：《生物医学工程学杂志》2010年第2期369-372,共4页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(30570693);四川省科技厅攻关计划科研基金资助项目(2006Z09-030)

摘　　要：本研究旨在构建可溶性血管内皮生长因子(vascular endothelial growth factor,VEGF)受体1(soluble fmslike tyosine kinase-1,sFlt-1)的真核表达质粒pcDNA3.1-sFlt-1,并观察sFlt-1对血管内皮细胞增殖的影响。提取人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)总RNA,扩增Flt-1基因胞外1-3结构域,构建真核表达质粒pcDNA3.1-sFlt-1,测序鉴定基因序列。将重组质粒转染Lewis肺癌细胞,采用RT-PCR和SDS-PAGE检测sFlt-1在基因及蛋白水平的表达情况。MTT法检测sFlt-1对VEGF诱导的HUVECs生长的影响。结果显示:①插入片段序列正确;②sFlt-1在基因水平成功表达且转染后的Lewis肺癌细胞能分泌表达sFlt-1;③含sFlt-1的细胞上清液可明显抑制VEGF诱导的HUVECs增殖。本研究成功构建了真核表达质粒pcDNA3.1-sFlt-1,sFlt-1,在基因和蛋白水平均获得有效表达,且表达的蛋白可明显抑制由VEGF诱导的血管内皮细胞增殖。This study sought to construct recombinant eukaryotic plasmid pcDNA3.1-sFlt-1 and observe its effect on proliferation of vascular endothelial cells.Total RNA was extracted from human umbilical vein endothelial cells（HUVECs） firstly.The 1st-3rd Ig-like domains of Flt were amplified by polymerase chain reaction（PCR） from the full-length cDNA.Subsequently,the PCR product was cloned into the eukaryotic plasmid pcDNA3.1（＋）/myc-His.The constructed recombinant plasmid pcDNA3.1-sFlt-1 was sequenced.Then recombinant plasmid was transfected into Lewis lung cancer cells.RT-PCR and SDS-PAGE were used to detect the expression of soluble vascular endothelial growth factor（VEGF） receptor mRNA and protein,respectively.MTT method was used to evaluate the effect of sFlt-1 protein on proliferation of HUVECs induced by VEGF.The results showed： ①The sequence of inserted fragment was correct.②Lewis lung cancer cells with recombinant plasmid transfection could express the soluble VEGF receptor mRNA and protein stably.③Culture supernatant of Lewis lung cancer cells with sFlt-1 could significantly inhibit the proliferation of HUVECs induced by VEGF.These data suggested that recombinant eukaryotic plasmids pcDNA3.1-sFlt-1 was constructed successfully,sFlt-1 mRNA and protein were expressed in eukaryotic system stably and sFlt-1 protein could significantly inhibit the proliferaton of endothelial cells induced by VEGF.

关 键 词：血管内皮生长因子 可溶性血管内皮生长因子受体1 内皮细胞 真核表达 抗血管生成 

分 类 号：Q786[生物学—分子生物学]