本文選題：人髓樣相關(guān)蛋白8　切入點：膿毒癥　出處：《南方醫(yī)科大學(xué)》2010年碩士論文

【摘要】：人髓樣相關(guān)蛋白8 (myeloid-related protein-8,MRP8)是鈣結(jié)合蛋白S100家族成員之一,具有EF手型結(jié)構(gòu)。MRP8相對分子質(zhì)量比較小,其單體由2個EF手Ca2+結(jié)合區(qū)和與之相連的中央鉸鏈區(qū)組成,形成螺旋-環(huán)-螺旋(Helix-Loop-Helix, H-L-H)結(jié)構(gòu)。其中位于C端的EF手型基序與Ca2+親和力較高,與Ca2+結(jié)合后,該蛋白的構(gòu)象發(fā)生改變,暴露出與靶蛋白相互作用的位點,從而發(fā)揮相關(guān)的生物學(xué)功能。MRP8最早發(fā)現(xiàn)于中性白細(xì)胞中,被認(rèn)為是一種具有免疫原性的蛋白。隨著研究的深入,發(fā)現(xiàn)該蛋白與炎癥高度相關(guān),能夠募集單核細(xì)胞至炎癥灶處,是許多急性和慢性炎癥的重要促炎因子。另外MRP8／MRP9形成異二聚體在炎癥中起了重要作用,該蛋白的血清濃度與炎癥活動有關(guān),因此,臨床上常用其作為監(jiān)測疾病發(fā)展變化及治療愈后的指標(biāo)。 脂多糖(lipopolysaccharide, LPS)在革蘭氏陰性菌感染的炎癥反應(yīng)中起到了至關(guān)重要的作用。該細(xì)菌產(chǎn)物能夠引起包括巨噬細(xì)胞在內(nèi)的機體免疫系統(tǒng)產(chǎn)生特異性反應(yīng)。LPS介導(dǎo)的失控性機制的爆發(fā)會導(dǎo)致膿毒癥或者是全身炎癥反應(yīng)綜合癥,而后者是一個非傳染性的疾病。盡管采取了大量抗菌治療,但仍然是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LPS等激活的單核／巨噬細(xì)胞和內(nèi)皮細(xì)胞會大量釋放MRP8,同時壞死組織細(xì)胞崩解也釋放出大量的MRP8。釋放到細(xì)胞外的MRP8分子通過進(jìn)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,MRP8在炎癥反應(yīng)過程中起著重要的作用。 盡管目前對于MRP8的生物學(xué)功能尚未完全闡明,但是研究發(fā)現(xiàn)MRP8蛋白在炎癥及腫瘤中的高表達(dá)并具有細(xì)胞因子樣的效應(yīng),提示該蛋白在許多與炎癥及腫瘤發(fā)生發(fā)展過程中具有重要的作用。因而MRP8參與炎癥、應(yīng)激等反應(yīng)的分子基礎(chǔ),特別是作用于細(xì)胞相關(guān)信號過程成為近年來研究的熱點。并且發(fā)現(xiàn),MRP8能與細(xì)胞表面的硫酸肝素蛋白多糖(heparan sulfate proteoglycan, HSPG)及羧化聚糖發(fā)生相互作用。更重要的是研究發(fā)現(xiàn)CD36分子、晚期糖基化終末產(chǎn)物受體(receptor for advanced glycation end products, RAGE)可能是MRP8的細(xì)胞表面受體,然而,阻斷這些受體并不能完全抑制MRP8的胞內(nèi)信號傳遞,這提示細(xì)胞表面可能還存在其他的MRP8受體或信號轉(zhuǎn)導(dǎo)方式。結(jié)合近年來人們陸續(xù)發(fā)現(xiàn)一些細(xì)胞因子、生長因子或其受體能夠被內(nèi)化而參與胞內(nèi)的信號反應(yīng),我們推測MRP8也能夠被效應(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)。 內(nèi)吞是一個復(fù)雜并且高效的過程,細(xì)胞能夠攝取營養(yǎng)物質(zhì)并且與外界進(jìn)行聯(lián)系。內(nèi)吞小泡的形成過程非常復(fù)雜,首先外源性的蛋白有序的富集于細(xì)胞膜周圍,緊接著質(zhì)膜上的脂質(zhì)雙分子層發(fā)生巨大的變形重構(gòu)將蛋白包裹形成凹陷的吞噬小泡。內(nèi)化的過程不僅能夠從外界獲得物質(zhì)與能量,同時也能夠調(diào)節(jié)細(xì)胞表面某些受體的數(shù)量,包括信號通路受體、粘附分子和GPI偶聯(lián)受體。外源性物質(zhì)進(jìn)入細(xì)胞的方式主要有三種,經(jīng)典的依賴包涵素的內(nèi)吞途徑(clathrin-dependent endocytosis),即生物大分子通過與AP-2和其他的受體相互作用而進(jìn)入細(xì)胞。很多生物大分子,包括低密脂蛋白(low density lipoprotein,LDL)、轉(zhuǎn)鐵蛋白(transferrin)、表皮生長因子(epidermal growth factor, EGF)、胰島素等都是通過受體介導(dǎo)的內(nèi)吞作用內(nèi)化的。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。胞膜窖是一種表面分布著特定受體,富含膽固醇及特征性小窩蛋白(caveolin)的微結(jié)構(gòu)。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(cholera toxin, CTx)和猿病毒40 (simian virus 40, SV40)等的內(nèi)化過程。另一種包涵素非依賴的途徑,主要是一些與GPI受體家族結(jié)合的分子和糖脂類物質(zhì),這類物質(zhì)具有多形的結(jié)構(gòu)并且不具備堅固的外殼。硫酸肝素蛋白多糖是廣泛存在于細(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,本研究首先構(gòu)建MRP8與增強型綠色熒光蛋白(enhanced green fluorescent protein, EGFP)融合表達(dá)載體,利用共聚焦顯微鏡觀察MRP8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MRP8綠色熒光蛋白共同孵育,研究MRP8通過何種內(nèi)化通路進(jìn)行內(nèi)化;之后通過生物信息學(xué)的分析研究介導(dǎo)MRP8內(nèi)化的功能域；進(jìn)一步研究MRP8內(nèi)化后的去路問題。最后利用LiquiChip-液相芯片技術(shù)檢測MRP8經(jīng)刺激后細(xì)胞因子的分泌情況,探討MRP8內(nèi)化與炎癥的相關(guān)性,為后續(xù)進(jìn)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、MRP8以時間和能量依賴性方式先與胞膜結(jié)合,進(jìn)而形成內(nèi)吞小泡內(nèi)化進(jìn)入哺乳細(xì)胞。5min時,開始與細(xì)胞膜結(jié)合,1h左右達(dá)到平衡,9h基本代謝完全。第二、MRP8首先可能與細(xì)胞膜表面的HSPG上的HS鏈結(jié)合,并依賴其特異性受體,經(jīng)胞膜窖介導(dǎo)的內(nèi)吞途徑進(jìn)入細(xì)胞,而后細(xì)胞骨架參與了該蛋白的細(xì)胞內(nèi)活動；第三、MRP8的內(nèi)化必須依賴鈣離子,由兩個EF手型基序協(xié)同完成；第四、MRP8內(nèi)吞進(jìn)入細(xì)胞后,最終被泛素標(biāo)記進(jìn)入蛋白酶體降解。而該蛋白上第92位賴氨酸發(fā)生泛素化修飾是其降解的主要途徑。第五、MRP8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,是機體對抗炎癥的一項重要調(diào)節(jié)機制。 MRP8參與了全身炎癥反應(yīng)的過程,與許多炎癥及腫瘤有關(guān)。本研究對MRP8的內(nèi)化機制進(jìn)行了初步研究,且證實MRP8可能通過內(nèi)化降解,抑制其刺激細(xì)胞分泌炎癥因子的功能。這些研究結(jié)果不但加深了我們對MRP8生物學(xué)功能的認(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]:Human myeloid related protein 8 (myeloid-related protein-8 MRP8) is one of the members of the family of calcium binding protein S100, EF has the hand structure relative molecular mass of.MRP8 is relatively small, the monomer consists of 2 EF Ca2+ binding region and the central hinge connected with the area, the formation of helix loop - helix (Helix-Loop-Helix H-L-H) structure. Which is located in the C end of the EF hand motif Ca2+ with higher affinity, when combined with Ca2+, the protein conformation changes, exposed and target protein interaction sites, and thus play biological functions related to.MRP8 was first discovered in white blood cells, is considered as a kind of protein immunogenic. With further research, found the protein with inflammation is highly correlated to the recruitment of monocytes to inflammatory lesions, is an important proinflammatory factor of many acute and chronic inflammation. In MRP8 / MRP9 formation of two dimers in inflammation play The serum concentration of the protein is associated with inflammatory activity, so it is used clinically as a marker for monitoring the development and healing of the disease.
Lipopolysaccharide (lipopolysaccharide, LPS) on the inflammatory reaction of gram negative bacterial infection plays a very important role. The bacterial products can cause the body's immune system including macrophages, have lost control of the mechanism of specific reaction mediated by.LPS will lead to the outbreak of sepsis or systemic inflammatory response syndrome, which is a non infectious disease. Despite numerous antimicrobial treatment, but is still one of the major causes of death of critical patients, the mortality rate is as high as 30 to 70%. over the past ten years of clinical use of tumor necrosis factor (tumor necrosis, factor, TNF) and interleukin 1 (interleukin-1, IL-1) are not satisfactory antagonist treatment results. Recent studies show that inflammatory stimulation LPS activation of monocytes / macrophages and endothelial cells release a lot of MRP8 at the same time, necrotic tissue cell disintegration also release The release of MRP8 molecules of MRP8. released into the extracellular through further activation of monocytes or endothelial cells such as induced expression and release of inflammatory factors and adhesion molecules, including lead and participate in the brain, lung, gastrointestinal tract, joint inflammation, injury of heart and other organs and extensive systemic inflammatory response even death. Thus, MRP8 plays an important role in the inflammatory process.
Although the biological function of MRP8 has not been fully elucidated, but the study found that high expression of MRP8 protein in inflammation and tumor and has the effect of cytokine, which plays an important role in suggesting that the occurrence of the protein in many inflammatory and tumor development. Thus MRP8 is involved in inflammation, the molecular basis of stress reaction, especially the effect on the cell signaling process becomes a hotspot in recent years. And it is found that MRP8 with cell surface heparan sulfate proteoglycan (heparan sulfate, proteoglycan, HSPG) and carboxylation chitosan interaction. More important is the study found that CD36 molecules, advanced glycation end product receptor (receptor for advanced glycation end products, RAGE) is a cell surface receptor of MRP8, however, blocking these receptors can not completely inhibit MRP8 intracellular signaling, suggesting that cell surface There may be other MRP8 receptor or signal transduction. In recent years, people have found some combination of cytokines, growth factors or their receptors can be internalized in response to intracellular signaling, we hypothesize that MRP8 can also be the effect of cellular internalization, biological function and its internalization activity was closely related to the rich.
Endocytosis is a complex and efficient process, cells can intake of nutrients and contact with the outside world. The forming process is very complex endocytic vesicles, the exogenous protein enriched in the orderly around the cell membrane, followed by lipid bilayer membrane on the deformation of reconstruction of giant protein inclusion formation in depression the phagocytic vesicle internalization process. Not only can obtain the material and energy from the outside, but also can regulate the cell number of some surface receptors, including signaling receptors, adhesion molecules and GPI coupled receptors. There are mainly three kinds of exogenous substances into cells, the classical endocytic pathway dependent inclusion pigment (clathrin-dependent endocytosis), namely biological macromolecules with AP-2 and other receptor interactions into cells. Many biological molecules, including low density lipoprotein (low density, lipoprotein, LDL Transferrin (transferrin), epidermal growth factor (epidermal), growth factor, EGF), insulin is internalized by receptor-mediated endocytosis. The nonclassical endocytic pathway does not depend on the inclusion of one of the most important is the caveolar endocytosis (caveolae) mediated by the cell membrane. The cellar is a surface distribution of a specific receptor, rich in cholesterol and characteristic of caveolin-1 (caveolin) micro structure. The first study this structure plays an important role in the cell signal transduction process, discovered in recent years for its involvement in various bacteria and virus particles, such as cholera toxin (cholera toxin, CTx and simian virus 40 (Simian) virus 40, SV40) internalization process. Another clathrin dependent way, is the main molecular and glycolipids and substance GPI receptor family with the structure of this kind of material is pleomorphic and with no preparation solid protein heparan sulfate shell. Polysaccharide is a kind of glycoprotein exists widely in cell surface and basement membrane by heparin sulfate (heparan, sulfate, HS) and core protein covalently linked to activation of specific receptors as co receptor ligands in the regulation of many cells, mechanical support, adhesion, movement, proliferation, and plays an important role in the the differentiation and morphogenesis. In addition, the research also indicates that HSPG also plays an important role in the internalization process, may be an important molecular mechanism of the internalization of universal.
Based on the above understanding, this study constructed MRP8 and enhanced green fluorescent protein (enhanced green fluorescent protein, EGFP) fusion expression vector, using a confocal microscope to observe whether MRP8 has internalized activity; then use a series of related pathway inhibitors and internalization of fluorescent dyes and MRP8 green fluorescent protein were incubated on MRP8 by what kind of internalization pathway after internalization; through the bioinformatics analysis of MRP8 domain mediated internalization; further research on the way of MRP8 after internalizing problems. Finally using LiquiChip- liquid chip technology to detect MRP8 by secretion of cytokines after stimulation, investigate the internalization of MRP8 correlation with inflammation, lay the foundation for further study further.
Through the above research, we obtained the following conclusions. First, MRP8 binding and cell membrane in time and energy dependent manner, and the formation of endocytic vesicles internalized into mammalian cell.5min, began to combine with the cell membrane, 1H balance, 9h basic metabolism. Second, MRP8 may be the first surface and cell membrane the HSPG HS chain with, and depends on its specific receptor, enter the cell through the endocytic pathway of caveolae mediated, and cytoskeleton proteins involved in the intracellular activities; third, MRP8 internalization must rely on calcium, completed by two EF hand motif fourth, MRP8 collaboration; Swallow after entering the cell, eventually the ubiquitin tag into proteasomal degradation. The protein of ninety-second lysine ubiquitinated is the main pathway for its degradation. Fifth, internalization and proinflammatory effect of MRP8 is accomplished through two different signal pathways The purpose of internalization is to degrade and inhibit the continuous aggravation of inflammation, which is an important regulatory mechanism for the body against inflammation.
MRP8 is involved in the process of the systemic inflammatory response, many associated with inflammation and tumor. In this study, internalization mechanism of MRP8 was studied, and confirmed by MRP8 might inhibit the degradation of internalized function, stimulate cells to secrete inflammatory cytokines. These results not only deepen our understanding of the biological function of MRP8, but also may from the fundamental effects and changes in clinical sepsis and understanding and treatment of uncontrolled systemic inflammation, so it has very important theoretical basis and clinical significance.

【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】：2010
【分類號】：R363

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1 伍麗瓊;MRP8內(nèi)化的分子機制及其生物學(xué)功能研究[D];南方醫(yī)科大學(xué);2010年

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本文編號：1673221