發(fā)布時間：2018-07-05 19:42

  本文選題：神經(jīng)肽Y + 巨噬細胞�。� 參考：《第二軍醫(yī)大學》2012年博士論文

【摘要】：近年來的研究表明，炎癥反應在疾病譜中發(fā)病率和死亡率居前以及因病所致負擔嚴重的慢性疾病的發(fā)生、發(fā)展過程中起重要作用，如惡性腫瘤、心腦血管疾病、代謝性疾病和神經(jīng)退行性疾病等。機體對炎癥反應的調(diào)控機制精確而又復雜，涉及神經(jīng)、內(nèi)分泌和免疫系統(tǒng)的共同參與和調(diào)節(jié)。神經(jīng)(內(nèi)分泌)-免疫網(wǎng)絡在調(diào)節(jié)炎癥以及維持機體內(nèi)環(huán)境穩(wěn)定的過程中發(fā)揮了重要的作用。 神經(jīng)肽Y(Neuropeptide Y, NPY)是由36個氨基酸組成的多肽,1982年由瑞典的Karolinska學院的Tatemoto和Mutt從400kg豬腦組織中純化出來的。NPY廣泛分布于中樞和外周組織，尤以神經(jīng)系統(tǒng)的含量為高。NPY在中樞的主要作用包括對學習、記憶、攝食、生殖內(nèi)分泌等功能的影響。除了上述功能以外，NPY在哺乳動物免疫功能的調(diào)節(jié)方面以及在一些自身免疫性疾病發(fā)生發(fā)展中的作用也逐漸被揭示。在外周，交感神經(jīng)是NPY釋放的一大重要來源。有研究表明，外周交感神經(jīng)廣泛支配著外周免疫器官，，在一些局部組織，出現(xiàn)了交感神經(jīng)末梢和巨噬細胞形成的“類突觸”樣結(jié)構(gòu)，這為NPY直接調(diào)節(jié)局部炎癥以及巨噬細胞功能提供了生理解剖基礎。 巨噬細胞是機體固有免疫的重要組成細胞，由外周血單核細胞分化而來，幾乎分布于機體的各種組織中�；罨木奘杉毎ㄟ^分泌多種炎癥因子如TNF-α、IL-1β、IL-6、HMGB1以及大量炎性介質(zhì)如白三烯、前列腺素、彈性蛋白酶、溶菌酶、尿激酶等，加強局部炎癥反應。因此，在細胞和分子水平，明確NPY對巨噬細胞炎癥因子和小分子炎性介質(zhì)的調(diào)節(jié)作用和機制，是神經(jīng)免疫調(diào)節(jié)方面研究亟待解決的問題。 系統(tǒng)生物學是研究一個生物系統(tǒng)中所有組成成分(基因、mRNA、蛋白質(zhì)、代謝物等)的構(gòu)成，以及在特定條件下這些組分間相互關系的學科。代謝組學(metabonomics)是以組群指標分析為基礎，以高通量檢測和數(shù)據(jù)處理為手段，以信息建模與系統(tǒng)整合為目標的系統(tǒng)生物學的一個分支，是一種新的整體性的分析技術，它是研究生物體系受外部刺激所產(chǎn)生的所有代謝產(chǎn)物變化的科學，所關注的是代謝循環(huán)中分子量小于1000的小分子代謝物的變化，反映的是外界刺激或遺傳修飾的細胞或組織的代謝應答變化。因此，通過代謝組學技術，可以獲得NPY作用下巨噬細胞小分子炎性介質(zhì)的改變，明確其具體調(diào)節(jié)作用。蛋白質(zhì)是生物體內(nèi)功能的執(zhí)行者，生物體結(jié)構(gòu)和功能狀態(tài)的改變必然在蛋白組水平上表現(xiàn)出來。通過液相色譜聯(lián)合質(zhì)譜鑒定可以獲得關于蛋白質(zhì)組的變化信息，并通過生物信息學分析和文獻調(diào)研，尋找可能改變的信號通路或分子，為進一步闡述NPY的作用機理提供了高效的研究手段。 本課題以小鼠巨噬細胞為研究對象。首先，采用LPS刺激巨噬細胞造成炎性激活模型。在此基礎上，檢測NPY對巨噬細胞主要炎性因子分泌的影響，并通過代謝組學技術，研究NPY對巨噬細胞小分子炎性介質(zhì)分泌的影響。進一步，通過分別提取NPY作用下巨噬細胞胞核和胞漿部分的蛋白質(zhì)組，應用納升級二維液相色譜—串聯(lián)質(zhì)譜技術（2D-nano-LC-MS/MS）技術獲得關于蛋白質(zhì)組的變化信息，從中推測NPY的作用機制；針對蛋白質(zhì)組學得出的結(jié)果以及NPY可能的信號通路，研究NPY炎性調(diào)節(jié)的分子機制，具體內(nèi)容包括： 1.本文首先建立了LPS刺激巨噬細胞造成的炎癥模型，發(fā)現(xiàn)NPY能夠抑制巨噬細胞炎性因子TNF-α、IL-1β、IL-6的產(chǎn)生，但是有趣的是，我們發(fā)現(xiàn)能夠顯著提高巨噬細胞晚期炎癥因子HMGB1的分泌，而且該作用并非由于巨噬細胞壞死所導致的被動釋放，而是巨噬細胞的主動分泌所致。通過mRNA水平的檢測，我們發(fā)現(xiàn)NPY能夠快速提高HMGB1mRNA的轉(zhuǎn)錄，其較LPS的提高mRNA水平的時間更快。接下來，我們應用代謝組學技術檢測了LPS處理組合NPY+LPS處理組上清液中分子量小于1000的小分子代謝物的變化。主成分分析結(jié)果顯示，NPY處理改變了炎癥激活巨噬細胞的分泌模式。通過確認代謝產(chǎn)物類似分子離子后，我們發(fā)現(xiàn)NPY能夠顯著下調(diào)兩種炎癥介質(zhì)脂氧素A4（0.53倍）和前列腺素B2（0.54倍）的分泌。本文的研究結(jié)果顯示NPY能夠顯著抑制巨噬細胞炎癥因子TNF-α、IL-1β、IL-6的產(chǎn)生，能夠提高晚期炎癥因子HMGB1的主動分泌和mRNA水平。并且能夠改變炎癥激活巨噬細胞的分泌模式，下調(diào)小分子炎癥介質(zhì)脂氧素A4（0.53倍）和前列腺素B2（0.54倍）的分泌。 2. NPY能夠調(diào)節(jié)巨噬細胞炎癥因子和小分子炎性介質(zhì)的分泌。為了探索其中的分子機制，我們采用了基于2D-nano-LC-MS/MS的高通量蛋白組學研究技術，研究RAW264.7細胞在10-8mol/L處理后，在胞內(nèi)不同空間蛋白表達上呈現(xiàn)的差異，從而探索NPY炎性調(diào)節(jié)可能的信號途徑。我們發(fā)現(xiàn)經(jīng)過NPY處理后，RAW264.7細胞共有435個蛋白的表達呈現(xiàn)明顯差異。這些蛋白涵蓋了復雜的功能領域，包括能量代謝、細胞骨架、核酸代謝和炎癥功能等。我們選取了跟炎癥功能相關的蛋白進行探討。最后我們發(fā)現(xiàn)，NPY可能通過改變其受體介導內(nèi)吞過程中的連接分子β-arrestin2的空間分布發(fā)揮了炎癥調(diào)節(jié)作用，影響了部分炎癥因子的表達。并且從本實驗可以看出，基于2D-nano-LC-MS/MS的蛋白組學技術是信號通路研究的有效手段。 3. NPY在巨噬細胞上主要通過Ⅰ型受體發(fā)揮作用，該受體屬于Gq型蛋白耦聯(lián)受體。目前關于NPY作用的分子機制罕有報道。我們結(jié)合第一部分研究觀察到的炎性因子調(diào)節(jié)和第二部分的蛋白質(zhì)組研究。我們推測NPY對TNF-α調(diào)節(jié)是由于NPY內(nèi)吞過程中β-arrestin2分子向胞核轉(zhuǎn)運過程相關。在細胞水平我們發(fā)現(xiàn)NPY作用下β-arrestin2分子向胞核轉(zhuǎn)移。同時，我們發(fā)現(xiàn)NPY作用下，LPS引起的NF-κB p65亞基的入核量降低，說明NF-κB控制的炎癥轉(zhuǎn)錄過程受到了抑制。
[Abstract]:Recent studies have shown that inflammatory reactions play an important role in the development of chronic diseases, such as malignant tumors, cardiovascular and cerebrovascular diseases, metabolic diseases and neurodegenerative diseases, such as the incidence and mortality of the disease and the occurrence of serious chronic diseases caused by disease. It involves the joint participation and regulation of the nervous, endocrine and immune systems. The neural (endocrine) - immune network plays an important role in regulating inflammation and maintaining the stability of the body's body.
The neuropeptide Y (Neuropeptide Y, NPY) is a polypeptide of 36 amino acids. The.NPY distributed in the central and peripheral tissues from the Tatemoto and Mutt of the Karolinska College of Sweden in 1982 is widely distributed in the central and peripheral tissues, especially the main role of the nervous system in the central role of.NPY, including learning, memory, feeding, and reproduction. In addition to these functions, the role of NPY in the regulation of immune function in mammals and in the development of some autoimmune diseases is gradually revealed. In the peripheral, the sympathetic nerve is a major source of NPY release. In some local tissues, the "synapse like" like structure of the sympathetic nerve endings and macrophages has been developed, which provides a physiological anatomical basis for the direct regulation of local inflammation and macrophage function by NPY.
Macrophages are an important constituent of the body's inherent immunity, which are differentiated from peripheral blood mononuclear cells and are almost distributed in various tissues of the body. Activated macrophages are secreted by a variety of inflammatory factors such as TNF- alpha, IL-1 beta, IL-6, HMGB1, and a large number of inflammatory mediators such as Bai San, prostaglandins, elastase, lysozyme, urokinase, etc. Therefore, the regulation and mechanism of NPY on macrophage inflammatory factors and small molecular inflammatory mediators at the cellular and molecular level is an urgent problem to be solved in the study of neuroimmunomodulatory.
System biology is the study of the composition of all components in a biological system (gene, mRNA, protein, metabolite, etc.) and the interrelationship between these components under specific conditions. Metabonomics is based on the analysis of group index, using high flux detection and data processing as a means of information modeling and system. A branch of systematic biology that is integrated as a target. It is a new holistic analysis technique. It is a science to study the changes in all metabolic products produced by the external stimuli of the biological system. It is concerned with the changes in the small molecular metabolites of less than 1000 of the molecular weight in the metabolic cycle, reflecting the external stimulus or the fine genetic modification. The metabolic response of the cells or tissues changes. Therefore, by metabonomics, the changes in the small molecular inflammatory mediators of macrophages under the action of NPY can be obtained, and the specific regulatory effect is clear. Protein is the executor of the function of the organism. The changes in the structure and function of the organism must be displayed at the level of the protein group. The identification of protein groups can be obtained by spectral combined mass spectrometry. Through bioinformatics analysis and literature research, we can find signal pathways or molecules that may change. It provides an efficient means to further elaborate the mechanism of NPY's action.
This subject takes mouse macrophages as the research object. First, LPS stimulates macrophages to cause inflammatory activation model. On this basis, the effect of NPY on the secretion of main inflammatory factors of macrophages is detected, and the effect of NPY on the secretion of small molecular inflammatory mediators of macrophages is studied by metabonomics. Further, by extracting NPY, respectively. The protein groups of the nucleus and cytoplasm of macrophages were used to obtain the change information about the protein group by using the nano upgrading two-dimensional liquid chromatography tandem mass spectrometry (2D-nano-LC-MS/MS) technology, and to speculate on the mechanism of the action of NPY; the results of proteomics and the possible signaling pathway of NPY were used to study the regulation of NPY inflammatory regulation. The molecular mechanism, including the following:
1. this article first established an inflammatory model caused by LPS stimulating macrophages, and found that NPY could inhibit the production of macrophage inflammatory factors TNF- a, IL-1 beta, and IL-6, but interestingly, we found that we can significantly increase the secretion of late macrophage inflammatory factor HMGB1, and this effect is not due to the passive release of macrophage necrosis. It was caused by active secretion of macrophages. Through mRNA level detection, we found that NPY could rapidly increase the transcription of HMGB1mRNA. It was faster than LPS to increase mRNA level. Next, we detected a small molecular metabolite in the LPS treatment group of NPY+LPS treated group NPY+LPS treatment group with the molecular weight less than 1000 in the LPS treatment group. The results of principal component analysis showed that NPY treatment changed the secretion pattern of macrophages activated by inflammation. We found that NPY could significantly downregulate the secretion of lipoxygenin A4 (0.53 times) and prostaglandin B2 (0.54 times) in two kinds of inflammatory mediators by identifying the metabolites as molecular ions. The results of this study showed that NPY could be significantly inhibited. The production of macrophage inflammatory factor TNF- a, IL-1 beta, and IL-6 can improve the active secretion of advanced inflammatory factor HMGB1 and the level of mRNA, and can change the secretion pattern of macrophages activated by inflammation and reduce the secretion of lipoxygenin A4 (0.53 times) and prostaglandin B2 (0.54 times) in small molecular inflammatory mediators.
2. NPY can regulate the secretion of macrophage inflammatory factors and small molecular inflammatory mediators. In order to explore the molecular mechanism of these molecules, we used a high throughput protein group study technique based on 2D-nano-LC-MS/MS to explore the differences in the expression of different spatial proteins in the cell after 10-8mol/L treatment, thus exploring the NPY inflammation. We found that after NPY treatment, we found a significant difference in the expression of a total of 435 proteins in RAW264.7 cells. These proteins cover complex functional domains, including energy metabolism, cytoskeleton, nucleic acid metabolism, and inflammatory functions. We have selected the proteins related to the function of the phlogistic disease. Now, NPY may play an inflammatory regulatory role by changing the spatial distribution of its receptor in endocytosis, which affects the expression of some inflammatory factors. And it can be seen from this experiment that the proteomics technology based on 2D-nano-LC-MS/MS is an effective means of signaling pathway research.
3. NPY plays a role mainly through the type I receptor on macrophages. The receptor belongs to the Gq type protein coupled receptor. The current molecular mechanism of the action of NPY is rare. We have studied the regulation of inflammatory factors and the second part of the proteome study combined with the first part. We speculate that the NPY regulation of TNF- alpha is due to the endocytosis of NPY. We found that the beta -arrestin2 molecules were transferred to the nucleus under the action of NPY at the cellular level. At the same time, we found that under the action of NPY, the nucleation of the NF- kappa B p65 subunit induced by LPS was reduced, indicating that the transcriptional process controlled by NF- kappa B was inhibited.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363

【引證文獻】

相關博士學位論文 前1條

1 石瑞麗;瓜子金皂苷己抑制神經(jīng)細胞缺血再灌注損傷及抗神經(jīng)炎癥作用的體外研究[D];內(nèi)蒙古農(nóng)業(yè)大學;2013年

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