脂多糖结合蛋白抑制肽对内毒素诱导的U937细胞和内毒素血症小鼠的作用及机制研究

发布时间：2018-02-11 17:16

本文关键词：脂多糖脂多糖结合蛋白脂多糖结合蛋白抑制肽炎症抗炎 U937细胞钟形受体4 CD_(14) 核转录因子kappa B 肿瘤坏死因子alpha 一氧化氮小鼠内毒素血症肺泡巨噬细胞流式细胞检测分析逆转录-聚合酶链反应免疫组织　出处：《第三军医大学》2005年博士论文　论文类型：学位论文

【摘要】：内毒素又称脂多糖(lipopolysaccharide,LPS),是革兰阴性细菌外膜的主要成分。内毒素血症及与其有关的急性肺损伤(acute lung injury,ALI)、急性呼吸窘迫综合征(acute respiratory dysfunction syndrome,ARDS)和多器官功能障碍综合征(multiple organ dysfunction syndrome,MODS)是病人感染死亡的重要原因。脂多糖结合蛋白(LPS-binding protein,LBP)在炎症中具有双刃剑的作用,一方面,当LBP 的致炎位点与LPS 的类脂A 结合时,表现为信号跨膜转导,核转录因子激活,炎症介质生成,导致过激炎症反应发生;另一方面,当LBP 的抗炎位点与LPS 的类脂A 结合时,LBP将LPS 传递给脂蛋白或靶细胞,使LPS 被中和或者被靶细胞清除,表现为抗炎作用。因为LBP 作用的双重性,应用LBP 或抗LBP 抗体治疗内毒素血症均有一定的局限性,所以若仅阻断LBP 的致炎作用、保留其抗炎作用,则能提高内毒素血症的治疗效果。我们实验室用噬菌体随机12 肽库筛选获得了可与LBP 竞争结合LPS 的噬菌体克隆,其核心序列与LBP 的91～102 位氨基酸(WKVRKSFFKLQG)有明显同源性,推测该位点为LBP 的致炎位点。应用FMOC 固相法合成12 肽WKVRKSFFKLQG-NH2,即为LBP 抑制肽(简称P12)。推测LBP 抑制肽能与LBP 的致炎位点竞争结合LPS,从而起抗炎作用。基于上述假说和以前的研究基础,我们进一步就LBP 抑制肽对内毒素诱导的U937 细胞和内毒素血症小鼠的作用及机制进行探讨。一、实验方法:本实验包括体内和体外两部分。 1.体外试验:以人单核巨噬细胞株U937 细胞为研究对象。根据实验设计方案,U937 细胞被分为对照组、LPS 组(分为100 ng/ml 的小剂量组和1μg/ml 的大剂量组)及P12 组(分为小、中、大剂量组)。 (1) 夹心ELISA 检测P12 与LPS 的相对亲和力。 (2) 流式细胞检测分析(FACS)异硫氰酸荧光素标记的LPS(FITC-LPS)与U937细胞的结合。 (3) RT-PCR 和Western blotting(WB)检测U937 细胞膜受体CD_(14) 和跨膜受体钟
[Abstract]:Also called endotoxin lipopolysaccharide (lipopolysaccharide, LPS), is a major component of the outer membrane of gram negative bacteria. Endotoxemia and related acute lung injury (acute lung, injury, ALI), acute respiratory distress syndrome (acute respiratory dysfunction syndrome, ARDS) and multiple organ dysfunction syndrome (MODS multiple organ dysfunction syndrome) is an important cause of death in patients with infection. Lipopolysaccharide binding protein (LPS-binding protein LBP) is a double-edged sword in the role of inflammation, on the one hand, when the LBP induced inflammation and LPS lipid A binding sites, for signal transduction and activation of nuclear transcription factors, inflammatory mediators, resulting in excessive inflammatory reaction; on the other hand, when the inflammation site and LPS lipid A LBP binding, LBP LPS will be transferred to the lipoprotein or target cells, the LPS is neutralized or removed as target cells, anti Inflammatory effect. Because of the double role of LBP, the application of LBP or anti LBP antibody treatment of endotoxemia have certain limitations, so if only LBP blocking proinflammatory effect, retains its anti-inflammatory effect, can improve the therapeutic effect of endotoxemia. Our laboratory using phage random 12 peptide library screened phage clones can be LPS the combination of LBP and its core competition, 91~102 amino acid sequence with LBP (WKVRKSFFKLQG) has obvious homology, speculated that the LBP allele was inflammatory sites. The application of FMOC solid phase synthesis of 12 peptide WKVRKSFFKLQG-NH2, namely LBP inhibitory peptide (P12). We speculated that LBP inhibitory peptides can LPS binding and LBP induced inflammation site competition, and anti-inflammatory effect. Based on the above hypothesis and based on the previous, we further LBP inhibition and mechanism of peptide on endotoxin induced U937 cells and the role of endotoxemia in mice Discuss.
1. Experimental methods: this experiment includes two parts of the body and in vitro.
1. in vitro test: human mononuclear macrophage cell line U937 cells as the research object. According to the experimental design, U937 cells were divided into control group, LPS group (divided into 100 ng/ml low dose group and 1 g/ml large dose group) and P12 group (divided into small, medium, high-dose group).
(1) the relative affinity of P12 and LPS was detected by sandwich ELISA.
(2) flow cytometry analysis (FACS) the binding of LPS (FITC-LPS) labeled with fluorescein isothiocyanate to U937 cells.
(3) RT-PCR and Western blotting (WB) detection of U937 cell membrane receptor CD_ (14) and transmembrane receptor clock

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R363

【引证文献】