摘要
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。
Objective：To construct eukaryotic expression vector of hlL-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 OVCAR-3 cells by RT-PCR, ELISA and Western blot assay. The proliferative effect of IL-8 on OVCAR-3 cells was observed for 6 days by MTr. Meanwhile, the cell cycle was analyzed by FCM. Results： Agarose gel electrophoretic analysis of double digested.products of recombinant plasmid showed that duplicate DNA bands were seen clearly in the right location. DNA sequencing showed that the exon of the fragment matched the gene order reported in GenBank, and the reading frame remained constant. There was significant difference in the level of mRNA and protein between the recombinant plasmid transfected group and other control groups by RT-PCR, ELISA and Western blot assay （ all P 〈 0.05）. MTr showed that the optical density value（ D410 ） of the transfected group after 3 days was higher than that of other control groups. The FCM analytic result showed that IL-8 cDNA could promote the growth of OVCAR-3 cells by increasing the S-phase cell number and upgrading the proliferation index（ compared with the other groups P 〈 0.05 ）. Conclusion： The human interleukin 8 gene is successfully subcloned into eukaryotic expression vector and expressed in OVCAR-3 cells, which confirmed the promotion of OVCAR-3 cells by IL-8 through autocrine.
出处
《军事医学科学院院刊》
CSCD
北大核心
2008年第2期144-147,共4页
Bulletin of the Academy of Military Medical Sciences
基金
河北省医学科研课题(No.05013)
河北省强势特色学科基金
