膜表面核仁素对脂多糖所致TNF-α和IL-1β表达的影响  

Role of cell-surface nucleolin in lipopolysaccharide-stimulated expression and secretion of TNF-α and IL-1β

作　　者：方立[1,2] 王慷慨[1] 蒋磊[1] 蒋碧梅[1] 韦星[1] 宋岚[1] 邓恭华[1] 肖献忠[1] 

机构地区：[1]中南大学湘雅医学院病理生理学系休克研究室,长沙410078 [2]中南大学湘雅医院心内科,长沙410008

出　　处：《中南大学学报（医学版）》2008年第11期999-1004,共6页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金重点项目(30330280)~~

摘　　要：目的:观察人THP-1细胞膜表面核仁素对脂多糖(LPS)所致TNF-α和IL-1β表达的影响。方法:采用免疫荧光、免疫印迹等方法观察核仁素在THP-1细胞膜表面表达。利用抗体封闭策略使膜表面核仁素失活,以LPS刺激THP-1细胞的炎症细胞模型,采用逆转录PCR和酶联免疫吸附实验观察炎症因子TNF-α和IL-1β的表达与分泌。结果:免疫荧光结果显示核仁素呈斑点状位于细胞膜表面;免疫印迹检测显示核仁素和Fas存在于THP-1细胞膜蛋白中。逆转录PCR结果显示LPS(1000μg/L)处理1,2,3,4h后,IL-1β和TNF-α mRNA均表达上调,而核仁素抗体封闭明显抑制二者的表达。酶联免疫吸附检测发现,LPS(1mg/L)刺激4,12,24h后,促进THP-1细胞分泌TNF-α和IL-1β,而核仁素抗体封闭显著抑制LPS所致的炎症因子释放(P<0.05)。结论:THP-1细胞膜表面核仁素参与介导LPS所致早期炎症介质IL-1β和TNF-α的表达与分泌。Objective To explore the role of cell-surface nucleolin in lipopolysaccharide （LPS）-stimulated expression and secretion of TNF-α and IL-1β in human THP-1 monocytes. Methods Immuno-fluoreseence assay and Western blot were used to identify the expression of nu- cleolin on the surface of THP-1 monocytes. Inactivation of nucleolin was induced by anti-nucleolin monoclonal antibody blockage, and the expression and secretion of TNF-α and IL-1β were observed by using reverse transcription polymerase chain reaction （RT-PCR）and enzyme linked immuno-sorbent assay （ELISA）respectively in LPS-mediated human THP-1 monoeyte inflammatory model. Results Immuno-fluorescence showed that nucleolin was localized on the cell surface of THP-1 monocytes as indicated by dotted red fluorescence. Western blot assay indicated that nucleolin existed in the cell membrane fractions. RT-PCR assay showed that the expressions of TNF-α and IL-1β mRNA significantly increased at 2 h and 3 h after the treatment with 1 000μg/L LPS. After 1 h pretreatment with anti-nucleolin antibody, the levels of TNF-α and IL-1β mRNA decreased compared with an anti-nucleolin antibody untreated group and an irrelevant IgG ＋ LPS group （ P 〈 0.05 ）. ELISA assay showed that the pretreatment with anti-nucleolin antibody inhibited significantly the secretion of LPS-induced levels of TNF-α and IL-1β after 4, 12 and 24 h treatment with 1 000 μg/L LPS. Conclusion Nucleolin expresses on the cell surface of THP-1 monocytes and involves in the LPS- mediated expression and secretion of TNF-αand IL-1β.

关 键 词：核仁素 THP-1单核细胞 脂多糖 炎症因子 基因表达 

分 类 号：R363[医药卫生—病理学]