本文關(guān)鍵詞：利用親和磷酸蛋白組學篩選和鑒定氧化應(yīng)激重塑型LPS信號通路中的新信號分子　出處：《第一軍醫(yī)大學》2006年博士論文　論文類型：學位論文

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【摘要】： 前期研究發(fā)現(xiàn)，缺血／再灌注損傷產(chǎn)生的氧化應(yīng)激可以引發(fā)機體對第二刺激物(通常為細菌脂多糖)產(chǎn)生強烈得多的反應(yīng)，從而使得病人更容易發(fā)生多器官功能衰竭綜合征，此即為所謂的“兩次打擊”理論。迄今為止，盡管有相當多的研究針對氧化應(yīng)激誘導的信號通路或經(jīng)典的LPS信號通路在感染中的作用作了相應(yīng)探討，然而，這些研究很少涉及氧化應(yīng)激和LPS信號通路的相互增效效應(yīng)。實際上，在預先存在氧化應(yīng)激的狀態(tài)下，細胞內(nèi)的LPS信號通路和傳統(tǒng)意義上的LPS信號通路有很大區(qū)別。體內(nèi)和體外研究均證實，氧化應(yīng)激狀態(tài)可以引發(fā)巨噬細胞針對LPS刺激分泌更多的細胞因子和趨化因子，從而導致過度的免疫反應(yīng)。此外，，不同于傳統(tǒng)LPS信號通路，這種激活作用應(yīng)歸因于src／PI-3K介導的超量NF-kappa B核轉(zhuǎn)位。 據(jù)此，先前的文獻提出了“氧化應(yīng)激可以導致細胞內(nèi)LPS信號通路發(fā)生重塑”這一理論，然而，由于對參與這種重塑型LPS信號通路的信號分子知之甚少，我們難以準確地闡明其機制。 迄今為止，還沒有文獻報道系統(tǒng)地比較傳統(tǒng)LPS信號通路和氧化應(yīng)激下發(fā)生重組的LPS信號通路的差別。為了更加全面和系統(tǒng)地研究氧化應(yīng)激狀態(tài)下的LPS信號通路，本文以PMA誘導分化的人外周血Thp-1巨噬細胞為模型，比較LPS刺激的細胞在有或無氧化應(yīng)激提前處理的情況下磷酸化蛋白質(zhì)組的差異，力圖篩選更多潛在的信號分子。通過BD公司的PMAC磷酸化蛋白純化柱，本研究富集了高達400μg磷酸化蛋白，與此同時，改良的分級染色法大大提高了檢測靈敏度。經(jīng)過這些方法的優(yōu)化，我們最終在雙向電泳圖譜上檢測到了大約1150個磷酸化蛋白斑點，遠遠超過此前報道的約700個，增加了低豐度的調(diào)節(jié)分子被檢出的可能性。 相對于僅用LPS刺激的Thp-1細胞，在有氧化應(yīng)激提前刺激的Thp-1細胞中，有29個磷酸化蛋白斑點在雙向電泳圖譜中表現(xiàn)出重復的差異，其中，16個斑點發(fā)生了上調(diào)(包括新出現(xiàn)的斑點)，11個發(fā)生了下調(diào)(包括消失的斑點)，另外的2個在雙向電泳圖譜中的位置坐標發(fā)生了變化。我們選擇了這些差異蛋白進行了質(zhì)譜分析，到目前為止鑒定出了其中5個，這些蛋白參與多種多樣的細胞過程例如蛋白質(zhì)降解、信號轉(zhuǎn)導和蛋白質(zhì)折疊等。其中，proteasome beta-4亞基在有氧化應(yīng)激時下調(diào)明顯，而核基質(zhì)蛋白NMP-238明顯上調(diào)。此前曾有報道表明蛋白酶體在不同水平上參與傳統(tǒng)LPS信號通路的調(diào)節(jié)：1，通過泛素-蛋白酶體通路負調(diào)節(jié)p38的活性；2，通過此通路負調(diào)節(jié)IRAK的活性；3，與LPS分子的直接結(jié)合，并通過糜蛋白酶活性將細菌抗原呈遞給MHC 1類分子。而核基質(zhì)蛋白NMP-238是激活Wnt／beta-catenin調(diào)控基因轉(zhuǎn)錄的一個關(guān)鍵輔助因子，而最近的研究也顯示W(wǎng)nt／beta-catenin介導一些感染過程。因此，結(jié)合前期的研究，我們提出以下假說，除src／PI-3K／NF-kappaB外，蛋白酶體和核基質(zhì)蛋白NMP-238也很有可能參與氧化應(yīng)激下LPS通路的重塑，主要體現(xiàn)在下列水平上：1，蛋白酶體活性急劇降低，從而導致p38活性強烈升高，刺激產(chǎn)生更多的黏附分子和細胞因子；2，急劇下降的蛋白酶體活性解除了對IRAK的負調(diào)控，從而引起無限制的IRAK激活，導致下游的JNK／AP-1和NF-kappaB通路廣泛激活；3，下調(diào)的蛋白酶體活性減少了細菌的抗原呈遞，從而降低了機體對細菌感染的獲得性免疫能力。4，上調(diào)的NMP-238通過Wnt／beta-catenin通路加劇了氧化應(yīng)激下LPS通路的炎癥反應(yīng)。綜上所述，以上多種因素協(xié)同作用，從而激發(fā)機體在氧化應(yīng)激條件下產(chǎn)生對LPS異常強烈的免疫反應(yīng)，并導致機體對細菌LPS的易感性大大增加。目前，我們已開始對蛋白酶體和核基質(zhì)蛋白NMP-238在氧化應(yīng)激性LPS信號通路中的作用進行深入的功能研究，以驗證上述假說是否成立。與此同時，我們正采取分泌組學和膜蛋白組學的策略以篩選更多的調(diào)節(jié)分子并隨之開展進一步研究。
[Abstract]:The preliminary study found that oxidative stress, ischemia / reperfusion injury can lead to the body of the second generation of stimuli (usually lipopolysaccharide) an exaggerated response, which makes the patients more prone to multiple organ failure syndrome, the so-called "two hit" theory. So far, despite the role LPS signal pathway of considerable research on the classical signal pathway or induced by oxidative stress in infection in the study, however, the mutual synergistic effect of these studies rarely involve oxidative stress and LPS pathway. In fact, in advance of the oxidative stress state, intracellular LPS signal pathway of LPS signal pathway and the traditional sense is very different. In vivo and in vitro studies have confirmed that oxidative stress can induce macrophage in LPS stimulated the secretion of cytokines and chemokines more In addition, it is different from the traditional LPS signaling pathway, which should be attributed to the Src / PI-3K mediated overdose NF-kappa B nuclear transposition.
Accordingly, previous literature has proposed the theory that oxidative stress can lead to remodeling of intracellular LPS signaling pathway. However, because of little knowledge of signal molecules involved in this remodeling LPS signaling pathway, it is difficult for us to elucidate its mechanism accurately.
So far, no LPS signal pathway of recombination between traditional LPS signal pathway and oxidative stress are reported in the literature systematically under the difference. In order to more comprehensive and systematic study of LPS signaling pathway under oxidative stress, the PMA induced differentiation of human peripheral blood Thp-1 macrophage cells as a model, LPS stimulation the cells in the presence or absence of oxidative stress in advance when processing differences of phosphoproteome, to screening more potential signaling molecules. Purified through phosphorylation of PMAC protein of BD, this study enriched up to 400 protein phosphorylation of G, at the same time, the improved graded staining method and improve detection sensitivity. Through the optimization of these methods, we finally in electrophoresis detected about 1150 phosphorylated protein spots, far more than previously reported about 700, increased regulation of low abundance The possibility of a molecule being detected.
Compared with only using LPS stimulated Thp-1 cells, oxidative stress in LPS stimulated Thp-1 cells, 29 phosphorylated protein spots showed repeated differences in electrophoresis which occurred by 16 spots (including new spots), 11 embryos (including the disappearance of the spots 2), changes in the electrophoresis in position coordinates. Also we chose these proteins of mass spectrometry, so far identified 5 of them, these proteins involved in various cellular processes such as protein degradation, signal transduction and protein folding. Among them, proteasome beta-4 the subunit was significantly down regulated in oxidative stress, and nuclear matrix protein NMP-238 was up-regulated. Earlier reports suggest that regulation of proteasome is involved in the traditional LPS signal pathway on different levels: 1, through the ubiquitin protein Enzyme pathway negatively regulates p38 activity; 2, through this pathway negatively regulates the activity of IRAK; 3, direct binding of LPS molecules, and bacterial antigens to MHC 1 molecules by chymotrypsin activity. The nuclear matrix protein NMP-238 is a key auxiliary factor activating Wnt / beta-catenin regulation of gene transcription. Recent studies also show that Wnt / beta-catenin mediated some infection process. Therefore, based on the previous research, we propose the following hypothesis, in addition to the Src / PI-3K / NF-kappaB, the proteasome and nuclear matrix protein NMP-238 is likely involved in oxidative stress remodeling under the LPS pathway, is mainly reflected in the following levels: 1, proteasome activity decreases sharply, resulting in strong p38 activity increased, stimulates the production of adhesion molecules and cytokines more; 2, the proteasome activity decreased sharply to relieve the negative regulation of IRAK, resulting in no The limitation of IRAK activation, resulting in JNK / AP-1 and NF-kappaB pathways downstream of extensive activation; 3, proteasome reduces bacterial antigen presentation, thereby reducing the body of bacterial infection acquired immunity.4, upregulation of NMP-238 through Wnt / beta-catenin pathway exacerbated inflammatory response of LPS pathway under oxidative stress. In summary, these factors synergy, so as to stimulate the immune response strongly on LPS under oxidative stress, and lead to susceptibility to bacterial LPS greatly increased. At present, we are beginning to function to further study the effect of proteasome and nuclear matrix protein NMP-238 in LPS signaling pathway in oxidative stress and to test this hypothesis is established. At the same time, we are taking secretome and membrane proteomics strategy to regulate molecular screening more and more Further research is carried out.

【學位授予單位】：第一軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R363

【參考文獻】

相關(guān)期刊論文 前1條

1 陳意生,史景泉;多器官功能障礙綜合征的病理基礎(chǔ)[J];創(chuàng)傷外科雜志;2001年01期

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