摘要
目的 研究过表达巨噬细胞移动抑制因子（MIF）对子宫颈癌SiHa细胞中白细胞介素8（IL-8）、基质金属蛋白酶9（MMP-9）表达及细胞侵袭迁移能力的影响.方法 化学合成MIF cDNA,设计含Xhol和BamHI酶切位点的引物序列,利用聚合酶链反应（PCR）方法 扩增MIF基因片段,构建人pEGFP-N1/MIF真核表达载体,采用脂质体转染法将载体转染到SiHa细胞中；酶联免疫吸附法（ELISA）检测各组上清液中MIF的表达,采用实时荧光定量PCR、免疫细胞化学法分别检测各组细胞中MIF、IL-8、MMP-9 mRNA和蛋白的表达,Boyden小室穿膜迁移实验检测过表达MIF对细胞迁移能力的影响.结果 转染pEGFP-N1/MIF组细胞上清液中MIF蛋白表达明显升高（F组别=8267.564,P＜ 0.01）；转染pEGFP-N1/MIF组细胞中MIF、IL-8、MMP-9 mRNA及蛋白也均过表达（F值分别为7019.619、2148.094、3303.540、1565.114、2807.300、523.466,均P＜0.01）；Pearson相关分析表明,MIF与IL-8、MMP-9之间mRNA和蛋白的表达均存在正相关（r值分别为0.865、0.895、0.934、0.908,均P＜0.01）,转染pEGFP-Nl/MIF组细胞的迁移能力明显增强（F=3430.898,P＜ 0.01）.结论 子宫颈癌SiHa细胞中过表达MIF能提高子宫颈癌细胞的侵袭、转移能力,其机制可能与IL-8、MMP-9表达上调相关.
Objective To investigate the effects of macrophage migration inhibitory factor （MIF） overexpression on the expression of interleukin-8 （IL-8）,martix metalloproteinase-9 （MMP-9） and invasion of human cervical cancer SiHa cells.Methods Chemical synthesis MIF eDNA gene,designed primer sequence including XhoI and BamHI enzyme sites,MIF gene was amplified by polymerase chain reaction （PCR）,constructed eukaryotic expression vector pEGFP-N1/MIF and transfected into SiHa cells using Lipofectamine and won over-expression of MIF.The expression of MIF in supernatant fluid was detected by ELISA,the expression of MIF,IL-8,MMP-9 in both mRNA and protein levels were detected by real-time fluorescence quantitative-PCR and immunocytochemistry respectively.The effect of over-expressed MIF on migration was detected by Boyden small chamber.Results The expression of protein in supernatant fluid transfected with pEGFP-N1/MIF was significantly increased （Fgroup =8267.564,P 〈 0.01）,the expression of MIF,IL-8,MMP-9 in both mRNA and protein in SiHa cells transfected with pEGFP-N1/MIF were significantly increased （F values were 7019.619,2148.094,3303.540,1565.114,2807.300,523.466,P 〈 0.01）,and there was a positive correlation among MIF,IL-8,MMP-9 expression in both mRNA and protein （r values were 0.865,0.895,0.934,0.908,P 〈 0.01）.Invasion ability in SiHa cells transfected with pEGFP-N1/MIF was obviously increased （F=3430.898,P〈 0.01）.Conclusion The over-expression MIF gene in SiHa cells can promote cervical cancer cell invasion and metastasis of ability,which could be associated with the upregulation of IL-8 and MMP-9 expression.
出处
《肿瘤研究与临床》
CAS
2013年第10期649-653,658,共6页
Cancer Research and Clinic
基金
山西省自然科学基金（2010011047-3）
关键词
巨噬细胞移动抑制因子
白细胞介素8
基质金属蛋白酶9
转染
子宫颈肿瘤
Macrophage migration inhibitory factor
Inrerleukin-8
Martix metalloproteinase-9
Transfection
Uterine cervical neoplasms
