IL-8基因真核表达载体的构建及其在OVCAR-3卵巢癌细胞中的表达

目的:构建白细胞介素8(interleukin 8,IL-8)基因真核表达载体,瞬时转染OVCAR-3卵巢癌细胞,为进一步探讨其与肿瘤发生发展关系奠定基础.方法:采用RT-PCR法自健康志愿者外周血单个核细胞扩增IL-8基因,构建pcDNA3.1( )/IL-8重组质粒;脂质体介导将外源基因转入OVCAR-3卵巢癌细胞,半定量RT-PCR、Western印迹及ELISA法鉴定其mRNA及蛋白瞬时表达后,MTT法检测细胞转染前后生长增殖特性的改变,FCM检测细胞周期的变化.结果:重组质粒双酶切电泳结果显示,相应位置(300 bp,5 400 bp)可见2条清晰特异的DNA条带;DNA测序结果进一步显示,重组质粒中插入的基因片段全长 300 bp,编码序列与GenBank报道的基因序列相符,阅读框保持不变;RT-PCR、Western 印迹检测及ELISA检测结果显示,转染后OVCAR-3细胞IL-8 mRNA及蛋白表达水平均明显升高(P<0.05);MTT检测结果显示,IL-8转染细胞组转染3 d后的光密度值(D值)显著高于未转染细胞组(P<0.05);FCM分析结果显示,OVCAR-3细胞转染IL-8基因后进入S期的细胞比例显著增高,增殖指数也相应上升,与未转染及空质粒转染组细胞相比,差异显著(P<0.05).结论:IL-8基因真核表达载体成功构建并瞬时转染OVCAR-3卵巢癌细胞;IL-8可以自分泌方式促进卵巢癌OVCAR-3细胞生长增殖.

Construction of human interleukin 8 gene eukaryotic expression vector and its expression in OVCAR-3 cells

Objective:To construct eukaryotic expression vector of hIL-8 and express it in OVCAR-3 cells after transient transfection in order to facilitate for further investigation of tumorigenesis and tumor growth.Methods:The IL-8 gene was amplified from human peripheral blood mononuclear cells of healthy volunteers by RT-PCR,and was cloned to eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid pcDNA3.1/hIL-8 was transfected into OVCAR-3 cells with Lipofectamine 2000.The expression of IL-8 was tested in...