本文選題：人髓樣相關(guān)蛋白14 + 膿毒癥　； 參考：《南方醫(yī)科大學(xué)》2009年碩士論文

【摘要】：人髓樣相關(guān)蛋白14(myeloid-related protein-14,MRP14)是鈣結(jié)合蛋白S100家族成員之一。MRP14相對分子質(zhì)量比較小,其單體由兩個EF手型基序組成,分別位于氨基末端和羧基末端,此結(jié)構(gòu)為鈣離子結(jié)合位點。其中,位于羧基端的EF手型基序與鈣離子結(jié)合力較強,與鈣離子結(jié)合后,其蛋白構(gòu)象發(fā)生改變,從而暴露出與靶蛋白的結(jié)合位點,通過與靶蛋白作用而發(fā)揮其生物學(xué)效應(yīng)。MRP14具有豐富的生物學(xué)效應(yīng),如參與蛋白質(zhì)的磷酸化,細(xì)胞的增殖與分化,細(xì)胞骨架構(gòu)建,調(diào)節(jié)細(xì)胞內(nèi)鈣離子的穩(wěn)態(tài)以及廣泛參與全身的炎癥反應(yīng)和組織損傷等。 膿毒癥仍然是目前臨床危重患者的重要死因之一,其死亡率高達(dá)30～70%。過去十幾年中臨床利用腫瘤壞死因子(tumor necrosis factor,TNF)和白細(xì)胞介素1(interleukin-1,IL-1)拮抗劑治療都未取得滿意效果。近年來的研究表明,致炎刺激脂多糖(lipopolysaccharide,LPS)等激活的單核/巨噬細(xì)胞和內(nèi)皮細(xì)胞會大量釋放MRP14,同時壞死組織細(xì)胞崩解也釋放出大量的MRP14。釋放到細(xì)胞外的MRP14分子通過進一步激活單核或內(nèi)皮等細(xì)胞,引起大量炎癥因子和黏附分子的表達(dá)和釋放,導(dǎo)致并參與了包括腦組織、肺、胃腸道、關(guān)節(jié)、心臟等多種臟器的炎癥損傷以及廣泛的全身性炎癥反應(yīng)甚至死亡。由此可見,MRP14在炎癥反應(yīng)過程中起著重要的中樞作用。 MRP14參與炎癥反應(yīng)的分子基礎(chǔ)及其相關(guān)信號過程近年來成為國內(nèi)外的研究熱點,也取得了一些進展,但很多過程仍不明確,特別是MRP14作用于細(xì)胞的信號機制。近年研究者們陸續(xù)鑒定出MRP14的一些細(xì)胞表面受體,包括晚期糖基化終末產(chǎn)物受體(receptor for advanced glycation end products,RAGE)、toll樣受體(toll-like receptor,TLR)家族成員等,但阻斷所有這些受體仍不能完全抑制MRP14對細(xì)胞的作用,表明細(xì)胞表面可能還存在其它受體或其它可能的信號轉(zhuǎn)導(dǎo)方式。MRP14在炎性細(xì)胞有大量表達(dá),結(jié)合近年來人們陸續(xù)發(fā)現(xiàn)一些細(xì)胞因子、生長因子或它們的受體可以被細(xì)胞內(nèi)化(internalization)而參與后續(xù)信號反應(yīng),我們推測MRP14可能也可以被效應(yīng)細(xì)胞內(nèi)化,且其內(nèi)化活性與其豐富的生物學(xué)功能密切相關(guān)。 細(xì)胞和外界進行著廣泛的物質(zhì)交換。除一些小分子物質(zhì)外,大多數(shù)生物大分子包括蛋白、多糖、多核苷酸以及一些病毒、細(xì)菌顆粒、壞死調(diào)亡細(xì)胞的產(chǎn)物都是通過內(nèi)化的方式進入細(xì)胞,內(nèi)化廣泛參與人體各種生理和病理過程。內(nèi)吞(endocytosis)是大分子物質(zhì)內(nèi)化的最重要的方式,是一個復(fù)雜的生物學(xué)過程,有多種信號通路參與,包含了廣泛的蛋白與蛋白以及蛋白與脂質(zhì)、糖類的相互作用。目前研究較清楚的特異性內(nèi)化方式是經(jīng)典的依賴包涵素的內(nèi)吞途徑(clathrin-dependent endocytosis),即生物大分子在相應(yīng)受體介導(dǎo)下通過形成有包涵素(clathrin)包被的小泡而被內(nèi)吞,很多生物大分子包括低密脂蛋白(lowdensity lipoprotein,LDL)、轉(zhuǎn)鐵蛋白(transferrin)、表皮生長因子(epidermalgrowth factor,EGF)、胰島素等都是通過受體介導(dǎo)的內(nèi)吞作用進行的。近年來研究較多的另一種重要的內(nèi)吞途徑是非經(jīng)典的不依賴包涵素的內(nèi)吞途徑,其中最重要的是胞膜窖(caveolae)介導(dǎo)的內(nèi)吞。胞膜窖是細(xì)胞膜上脂筏(lipid raft)的一種,是細(xì)胞膜上的一塊瓶頸形內(nèi)陷的膜結(jié)構(gòu),富含膽固醇、神經(jīng)鞘磷脂以及特征性蛋白—窖蛋白1(caveolin-1)。最初研究這個結(jié)構(gòu)在細(xì)胞信號轉(zhuǎn)導(dǎo)過程中起重要作用,近年發(fā)現(xiàn)其參與了多種細(xì)菌和病毒顆粒,如霍亂毒素(choleratoxin,CTx)和猿病毒40(simian virus 40,SV40)等的內(nèi)化過程。硫酸肝素蛋白多糖(heparan sulfate proteoglycan,HSPG)是廣泛存在于細(xì)胞表面和基底膜的一類糖蛋白,由硫酸肝素(heparan sulfate,HS)和核心蛋白共價連接而成,它們作為共受體調(diào)節(jié)許多配體的特異性受體的激活,在細(xì)胞的機械支持、粘附、運動、增殖、分化和形態(tài)形成中起重要作用。除此之外,研究還表明HSPG在內(nèi)化過程中也具有重要作用,可能是一種普遍的內(nèi)化機制中的重要分子。 基于以上認(rèn)識,本研究首先將MRP14與增強型綠色熒光蛋白(enhancedgreen fluorescent protein,EGFP)融合表達(dá),通過熒光定位觀察方法檢測MRP14是否具有內(nèi)化活性;接著利用一系列與內(nèi)化通路相關(guān)的抑制劑和熒光染料與帶有綠色熒光的MRP14蛋白共同孵育細(xì)胞,通過熒光共定位方法闡明MRP14通過何種內(nèi)化通路進行內(nèi)化;之后在生物信息學(xué)基礎(chǔ)上通過構(gòu)建MRP14上不同結(jié)構(gòu)域片斷與EGFP的融合表達(dá)載體,利用熒光顯微鏡研究介導(dǎo)MRP14內(nèi)化的功能域;進一步我們運用類似方法對MRP14內(nèi)化后的去路問題進行了研究;最后,我們利用liquichip-液相芯片技術(shù)檢測MRP14刺激后細(xì)胞因子的產(chǎn)生情況,從而初步探討MRP14內(nèi)化與炎癥的相關(guān)性,為后續(xù)進一步的深入研究打下基礎(chǔ)。 通過以上研究,我們得到了如下結(jié)論。第一、MRP14以時間和能量依賴性方式通過內(nèi)吞作用被哺乳動物細(xì)胞內(nèi)化,內(nèi)吞過程在15 min時已經(jīng)開始,1 h左右達(dá)到平衡,內(nèi)吞的MRP14顆粒散在分布于細(xì)胞質(zhì)內(nèi);第二、MRP14可能通過與細(xì)胞膜表面HSPG的HS鏈相互作用,并在其特異性受體協(xié)助下,通過依賴胞膜窖的內(nèi)吞途徑進入細(xì)胞,之后內(nèi)吞小泡在細(xì)胞骨架協(xié)助下在細(xì)胞內(nèi)運動,融合蛋白最終經(jīng)泛素標(biāo)記后,進入蛋白酶體降解;第三、MRP14的內(nèi)化必須在鈣離子存在的情況下,由兩個EF手型基序協(xié)同完成;第四、MRP14的內(nèi)化與促炎效應(yīng)是通過兩條不同的信號通路完成的,內(nèi)化的目的是為了降解,從而抑制炎癥的持續(xù)加重,是機體對炎癥反應(yīng)的一項重要調(diào)節(jié)機制。 本研究對MRP14的內(nèi)化機制進行了初步研究,且證實MRP14可能通過內(nèi)化降解,抑制其刺激細(xì)胞釋放炎癥因子的功能。這些研究結(jié)果不但加深了我們對MRP14生物學(xué)性質(zhì)的認(rèn)識,而且還有可能從根本上影響和改變臨床上對膿毒癥和失控性全身炎癥的認(rèn)識和治療方案,因而具有極為重要的基礎(chǔ)理論和臨床治療意義。
[Abstract]:The human myeloid related protein 14 (myeloid-related protein-14, MRP14) is one of the members of the calcium binding protein S100 family.MRP14 with relatively small relative molecular weight. The monomer consists of two EF hand types, which are located at the end of the amino group and the end of the carboxyl group. This structure is a calcium binding site. Among them, the EF hand motif at the carboxyl terminal and the calcium ion junction are located at the carboxyl terminus. After combining with calcium ion, the conformation of protein is changed and the binding site of the target protein is exposed. The biological effect of.MRP14 has a rich biological effect, such as the phosphorylation of protein, the proliferation and differentiation of cells, the construction of cytoskeleton, and the stability of intracellular calcium ion. State and extensive participation in systemic inflammatory response and tissue damage.
Sepsis is still one of the most important causes of death in critically ill patients at present. The mortality rate is up to 30 to 70%., and the clinical use of tumor necrosis factor (TNF) and interleukin 1 (interleukin-1, IL-1) antagonists in the past decade has not achieved satisfactory results. Recent studies have shown that inflammation stimulates lipopolysaccharide (lipopol). Ysaccharide, LPS) and other activated monocytes and macrophages and endothelial cells release a large number of MRP14, while necrotic tissue cells disintegrate and release a large number of MRP14. released to the extracellular MRP14 molecules by further activating mononuclear or endothelial cells, causing the expression and release of a large number of inflammatory factors and adhesion molecules, leading to and participating in the package. It can be seen that MRP14 plays an important central role in the process of inflammation, including the inflammatory damage of various organs such as brain tissue, lung, gastrointestinal tract, joint and heart, as well as extensive systemic inflammation and even death.
MRP14 participates in the molecular basis of the inflammatory response and its related signal process in recent years has become a hot spot at home and abroad, and some progress has been made, but many processes are still unclear, especially the signal mechanism of MRP14 on cell. In recent years, researchers have identified some cell surface receptors of MRP14, including late glycosylation end products. Receptor (receptor for advanced glycation end products, RAGE), toll like receptor (Toll-like receptor, TLR) family members, etc., but blocking all these receptors still can not completely inhibit the action of MRP14 to cells, suggesting that the cell surface may still have other receptors or other possible signal transduction pathways that have numerous tables in inflammatory cells. In recent years, a number of cytokines have been discovered, and growth factors or their receptors can be involved in cell internalization (internalization) to participate in subsequent signal reactions. We speculate that MRP14 may also be internalized in effector cells, and its internalization activity is closely related to its rich biological function.
In addition to some small molecular substances, most biological macromolecules include proteins, polysaccharides, polynucleotides, and some viruses, bacterial particles, and necrotic cells that enter cells through internalization, and are extensively involved in various physiological and pathological processes in the human body. Endocytosis (endocytosis ) is the most important way of macromolecular substance internalization. It is a complex biological process with a variety of signaling pathways involved, including a wide range of proteins and proteins, and the interaction of proteins with lipids and carbohydrates. The most clear specific internalization is the classic inclusion element endocytic pathway (clathrin-dependent endocy). Tosis), that is, biologic macromolecules are endocytosis by the corresponding receptor mediated vesicles that form inclusion (clathrin) inclusion. Many biological macromolecules include low density lipoprotein (lowdensity lipoprotein, LDL), transferrin (transferrin), epidermal growth factor (epidermalgrowth factor, EGF), insulin, etc. The other important endocytic pathway, which has been studied more recently, is an endocytic endocytic approach that is not dependent on inclusion elements. The most important is the endocytic endocytosis mediated by caveolae. The cell membrane cellar is a kind of lipid rafts (lipid raft) on the cell membrane. It is a membrane structure of a bottleneck on the cell membrane, which is rich in cholesterol. Nerve sphingomyelin and characteristic protein - caveolin 1 (caveolin-1). The initial study of this structure plays an important role in cell signal transduction. In recent years, it has been found to be involved in the internalization of a variety of bacteria and virus particles, such as cholera toxin (choleratoxin, CTx) and ape virus 40 (simian virus 40, SV40). Heparin sulfate polysaccharide (he) Paran sulfate proteoglycan, HSPG) is a kind of glycoprotein that widely exists on the surface of the cell and the basement membrane, which is covalently linked by heparin sulfate (heparan sulfate, HS) and core proteins. They are activated by the specific receptors regulating many ligands by common receptors, in cell mechanical support, adhesion, exercise, proliferation, differentiation and morphogenesis. In addition, the study also shows that HSPG plays an important role in internalization, and may be an important molecule in the internalization mechanism.
Based on the above understanding, this study first fused MRP14 with enhanced green fluorescent protein (enhancedgreen fluorescent protein, EGFP), and detected whether MRP14 had internalization activity by fluorescence localization observation, and then used a series of inhibitors and fluorescent dyes associated with internalized pathways and the MRP14 protein with green fluorescence. With the incubating cells, the internalization of MRP14 through the fluorescence co localization method is clarified, and then on the basis of bioinformatics, the fusion expression vector of different domain fragments on MRP14 and EGFP is constructed, and the functional domain of MRP14 internalization is studied by fluorescence microscopy. Further we use similar methods to internalize MRP14. The following problem is studied. Finally, we use liquichip- liquid phase chip technology to detect the production of cytokines after MRP14 stimulation, so as to preliminarily discuss the correlation between MRP14 internalization and inflammation, and lay a foundation for further further in-depth study.
Through the above study, we get the following conclusions. First, MRP14 is internalized in mammalian cells by endocytosis in a time and energy dependent manner. Endocytosis begins at 15 min and balances at about 1 h. The endocytic MRP14 particles are scattered in the cytoplasm; second, MRP14 may pass the HS chain to the cell membrane surface HSPG. Interaction, assisted by its specific receptor, enters cells by endocytosis pathway dependent on the cell membrane cellar, and then the endocytic vesicles move within the cell with the help of the cytoskeleton. The fusion protein is eventually labeled by ubiquitin and enters proteasome degradation; third, the internalization of MRP14 must be based on the presence of calcium ions by two EF hand groups. Fourth, the internalization and proinflammatory effect of MRP14 is accomplished through two different signaling pathways. The purpose of internalization is to degrade and inhibit the continuous aggravation of inflammation, which is an important regulatory mechanism for the body's inflammatory response.
This study has conducted a preliminary study on the internalization mechanism of MRP14, and confirmed that MRP14 may inhibit the release of inflammatory factors by its endogenous degradation. These results not only deepen our understanding of the biological properties of MRP14, but also have a fundamental effect on the clinical response to sepsis and out of control all over the body. The understanding and treatment of inflammation is of great importance in basic theory and clinical treatment.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R363

【參考文獻】

相關(guān)期刊論文 前2條

1 呂根法,衛(wèi)國,郭毅斌,肖光夏,鄭江,羅高興;多粘菌素B拮抗內(nèi)毒素的體外作用研究[J];第三軍醫(yī)大學(xué)學(xué)報;2004年14期

2 陳霞;李建生;;S100A9蛋白的生物學(xué)特性與臨床意義[J];國外醫(yī)學(xué)(內(nèi)科學(xué)分冊);2006年06期

，

本文編號：1915670