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  本文關鍵詞： 脂多糖 脂多糖結合蛋白 脂多糖結合蛋白抑制肽 炎癥 抗炎 U937細胞 鐘形受體4 CD_(14) 核轉錄因子kappa B 腫瘤壞死因子alpha 一氧化氮 小鼠 內毒素血癥 肺泡巨噬細胞 流式細胞檢測分析 逆轉錄-聚合酶鏈反應 免疫組織　出處：《第三軍醫(yī)大學》2005年博士論文　論文類型：學位論文

【摘要】：內毒素又稱脂多糖(lipopolysaccharide,LPS),是革蘭陰性細菌外膜的主要成分。內毒素血癥及與其有關的急性肺損傷(acute lung injury,ALI)、急性呼吸窘迫綜合征(acute respiratory dysfunction syndrome,ARDS)和多器官功能障礙綜合征(multiple organ dysfunction syndrome,MODS)是病人感染死亡的重要原因。脂多糖結合蛋白(LPS-binding protein,LBP)在炎癥中具有雙刃劍的作用,一方面,當LBP 的致炎位點與LPS 的類脂A 結合時,表現(xiàn)為信號跨膜轉導,核轉錄因子激活,炎癥介質生成,導致過激炎癥反應發(fā)生;另一方面,當LBP 的抗炎位點與LPS 的類脂A 結合時,LBP將LPS 傳遞給脂蛋白或靶細胞,使LPS 被中和或者被靶細胞清除,表現(xiàn)為抗炎作用。因為LBP 作用的雙重性,應用LBP 或抗LBP 抗體治療內毒素血癥均有一定的局限性,所以若僅阻斷LBP 的致炎作用、保留其抗炎作用,則能提高內毒素血癥的治療效果。我們實驗室用噬菌體隨機12 肽庫篩選獲得了可與LBP 競爭結合LPS 的噬菌體克隆,其核心序列與LBP 的91～102 位氨基酸(WKVRKSFFKLQG)有明顯同源性,推測該位點為LBP 的致炎位點。應用FMOC 固相法合成12 肽WKVRKSFFKLQG-NH2,即為LBP 抑制肽(簡稱P12)。推測LBP 抑制肽能與LBP 的致炎位點競爭結合LPS,從而起抗炎作用�；�于上述假說和以前的研究基礎,我們進一步就LBP 抑制肽對內毒素誘導的U937 細胞和內毒素血癥小鼠的作用及機制進行探討。 一、實驗方法:本實驗包括體內和體外兩部分。 1.體外試驗:以人單核巨噬細胞株U937 細胞為研究對象。根據(jù)實驗設計方案,U937 細胞被分為對照組、LPS 組(分為100 ng/ml 的小劑量組和1μg/ml 的大劑量組)及P12 組(分為小、中、大劑量組)。 (1) 夾心ELISA 檢測P12 與LPS 的相對親和力。 (2) 流式細胞檢測分析(FACS)異硫氰酸熒光素標記的LPS(FITC-LPS)與U937細胞的結合。 (3) RT-PCR 和Western blotting(WB)檢測U937 細胞膜受體CD_(14) 和跨膜受體鐘
[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

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R363

【引證文獻】

相關博士學位論文 前1條

1 魏麟;豬LBP和BPI基因多態(tài)性及其蛋白質N端功能研究[D];湖南農(nóng)業(yè)大學;2011年

相關碩士學位論文 前1條

1 胡莉;ApoA-I對LPS誘導的內毒素血癥小鼠的治療作用的研究[D];南華大學;2010年

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本文編號：1503577