神经肽Y对巨噬细胞炎性因子和小分子炎症介质的调节及机制研究

发布时间：2018-07-05 19:42

本文选题：神经肽Y + 巨噬细胞　；参考：《第二军医大学》2012年博士论文

【摘要】：近年来的研究表明，炎症反应在疾病谱中发病率和死亡率居前以及因病所致负担严重的慢性疾病的发生、发展过程中起重要作用，如恶性肿瘤、心脑血管疾病、代谢性疾病和神经退行性疾病等。机体对炎症反应的调控机制精确而又复杂，涉及神经、内分泌和免疫系统的共同参与和调节。神经(内分泌)-免疫网络在调节炎症以及维持机体内环境稳定的过程中发挥了重要的作用。神经肽Y(Neuropeptide Y, NPY)是由36个氨基酸组成的多肽,1982年由瑞典的Karolinska学院的Tatemoto和Mutt从400kg猪脑组织中纯化出来的。NPY广泛分布于中枢和外周组织，尤以神经系统的含量为高。NPY在中枢的主要作用包括对学习、记忆、摄食、生殖内分泌等功能的影响。除了上述功能以外，NPY在哺乳动物免疫功能的调节方面以及在一些自身免疫性疾病发生发展中的作用也逐渐被揭示。在外周，交感神经是NPY释放的一大重要来源。有研究表明，外周交感神经广泛支配着外周免疫器官，，在一些局部组织，出现了交感神经末梢和巨噬细胞形成的“类突触”样结构，这为NPY直接调节局部炎症以及巨噬细胞功能提供了生理解剖基础。巨噬细胞是机体固有免疫的重要组成细胞，由外周血单核细胞分化而来，几乎分布于机体的各种组织中。活化的巨噬细胞通过分泌多种炎症因子如TNF-α、IL-1β、IL-6、HMGB1以及大量炎性介质如白三烯、前列腺素、弹性蛋白酶、溶菌酶、尿激酶等，加强局部炎症反应。因此，在细胞和分子水平，明确NPY对巨噬细胞炎症因子和小分子炎性介质的调节作用和机制，是神经免疫调节方面研究亟待解决的问题。系统生物学是研究一个生物系统中所有组成成分(基因、mRNA、蛋白质、代谢物等)的构成，以及在特定条件下这些组分间相互关系的学科。代谢组学(metabonomics)是以组群指标分析为基础，以高通量检测和数据处理为手段，以信息建模与系统整合为目标的系统生物学的一个分支，是一种新的整体性的分析技术，它是研究生物体系受外部刺激所产生的所有代谢产物变化的科学，所关注的是代谢循环中分子量小于1000的小分子代谢物的变化，反映的是外界刺激或遗传修饰的细胞或组织的代谢应答变化。因此，通过代谢组学技术，可以获得NPY作用下巨噬细胞小分子炎性介质的改变，明确其具体调节作用。蛋白质是生物体内功能的执行者，生物体结构和功能状态的改变必然在蛋白组水平上表现出来。通过液相色谱联合质谱鉴定可以获得关于蛋白质组的变化信息，并通过生物信息学分析和文献调研，寻找可能改变的信号通路或分子，为进一步阐述NPY的作用机理提供了高效的研究手段。本课题以小鼠巨噬细胞为研究对象。首先，采用LPS刺激巨噬细胞造成炎性激活模型。在此基础上，检测NPY对巨噬细胞主要炎性因子分泌的影响，并通过代谢组学技术，研究NPY对巨噬细胞小分子炎性介质分泌的影响。进一步，通过分别提取NPY作用下巨噬细胞胞核和胞浆部分的蛋白质组，应用纳升级二维液相色谱—串联质谱技术（2D-nano-LC-MS/MS）技术获得关于蛋白质组的变化信息，从中推测NPY的作用机制；针对蛋白质组学得出的结果以及NPY可能的信号通路，研究NPY炎性调节的分子机制，具体内容包括： 1.本文首先建立了LPS刺激巨噬细胞造成的炎症模型，发现NPY能够抑制巨噬细胞炎性因子TNF-α、IL-1β、IL-6的产生，但是有趣的是，我们发现能够显著提高巨噬细胞晚期炎症因子HMGB1的分泌，而且该作用并非由于巨噬细胞坏死所导致的被动释放，而是巨噬细胞的主动分泌所致。通过mRNA水平的检测，我们发现NPY能够快速提高HMGB1mRNA的转录，其较LPS的提高mRNA水平的时间更快。接下来，我们应用代谢组学技术检测了LPS处理组合NPY+LPS处理组上清液中分子量小于1000的小分子代谢物的变化。主成分分析结果显示，NPY处理改变了炎症激活巨噬细胞的分泌模式。通过确认代谢产物类似分子离子后，我们发现NPY能够显著下调两种炎症介质脂氧素A4（0.53倍）和前列腺素B2（0.54倍）的分泌。本文的研究结果显示NPY能够显著抑制巨噬细胞炎症因子TNF-α、IL-1β、IL-6的产生，能够提高晚期炎症因子HMGB1的主动分泌和mRNA水平。并且能够改变炎症激活巨噬细胞的分泌模式，下调小分子炎症介质脂氧素A4（0.53倍）和前列腺素B2（0.54倍）的分泌。 2. NPY能够调节巨噬细胞炎症因子和小分子炎性介质的分泌。为了探索其中的分子机制，我们采用了基于2D-nano-LC-MS/MS的高通量蛋白组学研究技术，研究RAW264.7细胞在10-8mol/L处理后，在胞内不同空间蛋白表达上呈现的差异，从而探索NPY炎性调节可能的信号途径。我们发现经过NPY处理后，RAW264.7细胞共有435个蛋白的表达呈现明显差异。这些蛋白涵盖了复杂的功能领域，包括能量代谢、细胞骨架、核酸代谢和炎症功能等。我们选取了跟炎症功能相关的蛋白进行探讨。最后我们发现，NPY可能通过改变其受体介导内吞过程中的连接分子β-arrestin2的空间分布发挥了炎症调节作用，影响了部分炎症因子的表达。并且从本实验可以看出，基于2D-nano-LC-MS/MS的蛋白组学技术是信号通路研究的有效手段。 3. NPY在巨噬细胞上主要通过Ⅰ型受体发挥作用，该受体属于Gq型蛋白耦联受体。目前关于NPY作用的分子机制罕有报道。我们结合第一部分研究观察到的炎性因子调节和第二部分的蛋白质组研究。我们推测NPY对TNF-α调节是由于NPY内吞过程中β-arrestin2分子向胞核转运过程相关。在细胞水平我们发现NPY作用下β-arrestin2分子向胞核转移。同时，我们发现NPY作用下，LPS引起的NF-κB p65亚基的入核量降低，说明NF-κB控制的炎症转录过程受到了抑制。
[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.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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