IL-10对小鼠星形胶质细胞中IFN-γ诱导IDO表达调控机制

发布时间：2018-06-12 04:42

本文选题：吲哚胺2 + 3-双加氧酶　；参考：《电子科技大学》2014年硕士论文

【摘要】：背景:吲哚胺2,3-双加氧酶(IDO)是肝脏外唯一负责色氨酸代谢的限速酶,它可以降解必需氨基酸色氨酸并且生成一系列代谢产物。IDO活性的异常引起神经免疫调节异常,参与神经精神类疾病的发生。色氨酸代谢除了对免疫系统发挥作用,还能影响中枢神经系统(CNS)。IDO能够改变血清素的前体色氨酸进入犬尿氨酸代谢,导致一系列有毒代谢产物的生成。促炎性细胞因子可以激活IDO,进而激活色氨酸—犬尿氨酸途径,在CNS中,95%的色氨酸都是通过犬尿氨酸途径进行代谢。肿瘤坏死因子-α(TNF-α),干扰素-gamma(IFN-γ)和白介素-1β(IL-1β)等促炎性细胞因子以协同作用方式增强IDO活性和色氨酸代谢。而抗炎细胞因子白介素-4(IL-4)和白介素-10(IL-10)抑制T细胞、树突细胞和巨噬细胞中IDO介导的色氨酸分解代谢。但在CNS中,抗炎细胞因子对IDO的影响是不一致的。在神经内分泌细胞中,IL-10通过调节IDO表达可能提供保护作用,而抗炎细胞因子IL-4和白介素-13(IL-13)极大地增强了小胶质细胞中IDO的表达。目的:IL-10作为典型的抗炎细胞因子,不仅抑制炎症反应,也有神经保护功能,但是深层的机制并不清楚,是否IL-10通过调节色氨酸—犬尿氨酸途径而发挥功能以及星形胶质细胞中IL-10对IDO的作用仍待进一步研究。为了解决这个问题,本研究旨在探究在CNS中,是否IL-10在IDO-色氨酸代谢途径中作为一个潜在的调节介质发挥作用。方法:(1)取出生3天内的C57乳鼠大脑皮层,通过组织消化法所得星形胶质细胞,进行形态学观察、纯度鉴定。(2)采用星形胶质细胞培养方法,根据不同的处理,将原代培养的细胞分为四组:即a)空白对照组,b)IFN-γ处理组,c)IFN-γ+IL-10处理组,d)IL-10处理组。用免疫荧光染色(IF)、实时定量PCR(RT-PCR)、免疫蛋白印记(Western blot)方法来定量检测IDO的表达量变化。(3)利用免疫蛋白印记的方法检测信号转导与转录激活因子-3(STAT-3)磷酸化及下游细胞因子信号抑制因子(SOCS3)表达与IDO表达的相互作用,及其信号通路机制。并应用STAT-3抑制剂WP1066,探究IL-10发挥作用的分子机制,以便研究课题进一步深入。结果:实验结果表明,(1)通过机械振摇法得到的原代培养的星形胶质细胞纯度、存活率均达到95%以上,明显看出细胞分支明显,胞体伸展。(2)免疫荧光染、RT-PCR、western blot方法均显示,IFN-γ+IL-10处理组中IDO表达量显著低于IFN-γ处理组(P0.05)。(3)免疫荧光及Western blot结果显示,IL-10显著增强STAT-3磷酸化与SOCS3的表达(P0.05),明显抑制酪氨酸蛋白激酶1(JAK1)表达(P0.05)。应用STAT-3抑制剂WP1066后,显著降低IL-10对IDO表达的抑制作用,揭示了IL-10抑制IDO表达的分子机制。结论:(1)在原代星形胶质细胞中,IL-10明显减少IFN-γ诱导的IDO增多。(2)IL-10通过抑制JAK/STAT-1活性发挥这种作用。(3)STAT-3,SOCS3,JAK的活性表达是IL-10抑制IDO表达的内部信号通路。(4)在CNS疾病中,IDO表达并具有重要作用,探究IL-10对促炎性细胞因子IFN-γ引发的IDO表达的影响,可为在CNS中IL-10的神经保护作用提供理论依据与支撑,并为临床CNS疾病创立新的治疗靶点。
[Abstract]:Background: indolamine 2,3- dioxygenase (IDO) is the only speed limiting enzyme that is responsible for tryptophan metabolism outside the liver. It can degrade essential amino acid tryptophan and produce a series of abnormal.IDO activities of metabolites to induce abnormal neuromodulation and participate in the development of neuropsychiatric disorders. Tryptophan metabolism is not only effective in the immune system, but also in the immune system. It can also affect the central nervous system (CNS).IDO that can alter serotonin's protryptine into canine urinary ammonia metabolism, leading to the formation of a series of toxic metabolites. Pro-inflammatory cytokines can activate IDO and activate tryptophan - canine urinary ammonia pathway. In CNS, 95% of tryptophan is metabolized through the canine urinary ammonia pathway. Tumor necrosis factor - alpha (TNF- alpha), interferon -gamma (IFN- gamma) and interleukin -1 beta (IL-1 beta) and other inflammatory cytokines enhance IDO activity and tryptophan metabolism in synergistic manner, while the anti inflammatory cytokines interleukin -4 (IL-4) and interleukin -10 (IL-10) inhibit T cells, tree processes and macrophages, and tryptophan catabolism. In S, the effect of anti-inflammatory cytokines on IDO is inconsistent. In neuroendocrine cells, IL-10 may provide protection by regulating IDO expression, and anti-inflammatory cytokine IL-4 and interleukin -13 (IL-13) greatly enhance the expression of IDO in microglia. Objective: IL-10 is a typical anti-inflammatory cytokine, not only inhibiting the inflammatory response. It also has neuroprotective functions, but the underlying mechanism is not clear. Whether IL-10 plays the function by regulating tryptophan - canine urinary ammonia pathway and the role of IL-10 in astrocytes in IDO remains to be further studied. In order to solve this problem, the purpose of this study was to explore whether IL-10 was used in the IDO- tryptophan metabolic pathway in CNS. To play a role as a potential mediator. Methods: (1) taking the cerebral cortex of C57 rats of 3 days of birth, astrocytes obtained by tissue digestion, morphological observation and purity identification. (2) using astrocyte culture method, according to different treatment, the primary cultured cells were divided into four groups: a) blank control group, b) IF N- gamma treatment group, c) IFN- gamma +IL-10 treatment group, D IL-10 treatment group. Quantitative detection of IDO expression changes by immunofluorescence staining (IF), real-time quantitative PCR (RT-PCR), immunoglobulin imprint (Western blot) method. (3) detection of signal transduction and transcription activating factor phosphorylation and downstream cytokine signaling using immunoglobulin imprint The interaction between the expression of SOCS3 and the expression of IDO and its signaling pathway and the application of the STAT-3 inhibitor WP1066 to explore the molecular mechanism of the role of IL-10 in order to further study the subject. Results: the results showed that (1) the survival rate of the original cultured astrocytes obtained by the mechanical shaking method was reached. To more than 95%, it was obvious that the cell branches were obvious and the body extension. (2) immunofluorescence staining, RT-PCR, Western blot methods all showed that the IDO expression in IFN- gamma +IL-10 treatment group was significantly lower than that of IFN- gamma treatment group (P0.05). (3) the results of immunofluorescence and Western blot showed that IL-10 significantly enhanced STAT-3 phosphorylation and expression, obviously inhibiting cheese The expression of ammonia protein kinase 1 (JAK1) (P0.05). After the application of STAT-3 inhibitor WP1066, the inhibitory effect of IL-10 on the expression of IDO was significantly reduced and the molecular mechanism of IL-10 inhibition of IDO expression was revealed. Conclusion: (1) IL-10 significantly reduced IDO increase induced by IFN- gamma in the primary astrocytes. (2) IL-10 by inhibiting the activity to play this role. 3) the active expression of STAT-3, SOCS3 and JAK is the internal signal pathway of IL-10 inhibition of IDO expression. (4) in CNS disease, IDO expression is important to explore the effect of IL-10 on the expression of IDO of proinflammatory cytokine IFN- gamma, which can provide theoretical basis and support for the neuroprotective effect of IL-10 in CNS, and to create a new disease for clinical disease. Treat the target.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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