Fang Li, Wang Kang-kai, Jiang Lei, Jiang Bi-mei, Wei Xing, Song Lan, Deng Gong-hua, Xiao Xian-zhong
Laboratory of Shock, Department of Pathophysiology, Xiangya School of Medicine, Central South University, and Department of Cardiology, Xiangya Hospital, Changsha 410008, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2008 Nov;33(11):999-1004.
To explore the role of cell-surface nucleolin in lipopolysaccharide (LPS)-stimulated expression and secretion of TNF-alpha and IL-1beta in human THP-1 monocytes.
Immuno-fluorescence assay and Western blot were used to identify the expression of nucleolin 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-alpha and IL-1beta 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 monocyte inflammatory model.
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-alpha and IL-1beta mRNA significantly increased at 2 h and 3 h after the treatment with 1000 microg/L LPS. After 1 h pretreatment with anti-nucleolin antibody, the levels of TNF-alpha and IL-1beta 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-alpha and IL-1beta after 4, 12 and 24 h treatment with 1000 microg/L LPS.
Nucleolin expresses on the cell surface of THP-1 monocytes and involves in the LPS-mediated expression and secretion of TNF-alpha and IL-1beta.
探讨细胞表面核仁素在脂多糖（LPS）刺激人THP-1单核细胞表达和分泌肿瘤坏死因子-α（TNF-α）及白细胞介素-1β（IL-1β）中的作用。
采用免疫荧光法和蛋白质免疫印迹法鉴定核仁素在THP-1单核细胞表面的表达。通过抗核仁素单克隆抗体阻断诱导核仁素失活，在LPS介导的人THP-1单核细胞炎症模型中，分别采用逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）观察TNF-α和IL-1β的表达及分泌情况。
免疫荧光显示，核仁素定位于THP-1单核细胞的细胞表面，呈点状红色荧光。蛋白质免疫印迹分析表明核仁素存在于细胞膜组分中。RT-PCR分析显示，用1000μg/L LPS处理后2小时和3小时，TNF-α和IL-1β mRNA的表达显著增加。用抗核仁素抗体预处理1小时后，与抗核仁素抗体未处理组和无关IgG+LPS组相比，TNF-α和IL-1β mRNA水平降低（P<0.05）。ELISA分析表明，用抗核仁素抗体预处理可显著抑制在1000μg/L LPS处理4、12和24小时后LPS诱导的TNF-α和IL-1β水平的分泌。
核仁素在THP-1单核细胞的细胞表面表达，并参与LPS介导的TNF-α和IL-1β的表达及分泌。
