Tim-3对NLRP3炎性小体的调控作用及其机制研究

发布时间：2018-05-06 06:43

本文选题：Tim-3 + NLRP3炎性小体　；参考：《河南大学》2016年硕士论文

【摘要】：背景近年来,多种自身免疫性疾病及炎症性疾病如类风湿性关节炎、痛风、Ⅰ型糖尿病、胰腺炎、腹膜炎等发病率逐年升高,对人们生命健康造成了极大的威胁。研究表明,在多种自身免疫性疾病以及炎症性疾病的发病过程中存在NOD样受体蛋白3(NOD-like receptor,pyrin domain-containing 3,NLRP3)炎症小体的过度活化引起的炎症因子白细胞介素-1beta(inteleukine-1beta,IL-1β)大量释放,进而引起剧烈的炎症反应。所以,探索机体中NLRP3炎症小体的调控机制及寻找其负性调控分子成为了科研工作者迫切要解决的课题。目前研究认为,NLRP3炎症小体的活化需要两个信号:预激信号(第一信号)和激活信号(第二信号),其中第一信号主要负责NLRP3炎性小体复合体组分和效应分子的转录表达,第二信号则是将NLRP3炎性小体各组分由各自独立状态组合成复合体,同时激活NLRP3炎性小体,处于活化状态的NLRP3炎性小体可以促进炎症因子IL-1β、IL-18的成熟与释放,从而引起机体的炎症反应和特定条件下疾病的发生。T细胞免疫球蛋白及粘蛋白-3(T-cell Ig-mucin-3,Tim-3)是一新的具有抑制活性的免疫检查点分子。Tim-3在T细胞上的异常表达与多种免疫相关性疾病如肿瘤、病毒感染等相关,因此被认为是新一代的免疫调控靶点。近年来,Tim-3在天然免疫细胞上的表达及功能越来越受到人们的重视,然而Tim-3调控天然免疫的机制尚不十分清楚。作为天然免疫效应成分的炎症小体是否也受到Tim-3调控是非常值得探讨的课题。我们实验室在前期研究中发现Tim-3能够通过抑制TLR4参与调控炎症反应。鉴于TLR4在NLRP3炎性小体的活化过程中也发挥着重要的作用,那么Tim-3分子是否也参与调控NLRP3炎症小体的活化?其参与调控的机制是什么?这将是本课题研究的主要内容。目的探讨Tim-3对NLRP3炎症小体的调控作用及其机制。方法1.tim-3对nlrp3炎性小体活化作用的探讨。从tim-3高表达的转基因小鼠(tim-3-tg)和野生型小鼠(wt)中分离出腹腔巨噬细胞,使用nlrp3炎性小体活化的第一信号激活剂lps和第二信号激活剂atp进行刺激,观察巨噬细胞中nlrp3炎性小体的活化情况。使用tim-3融合蛋白(tim-3-ig)阻断鼠源巨噬细胞系raw264.7中tim-3信号通路,观察nlrp3炎性小体的活化情况;2.tim-3调控nlrp3炎性小体的机制探讨:对复合物成分转录及表达的影响。使用tim-3-ig阻断tim-3信号通路,观察鼠源巨噬细胞系raw264.7以及j774a.1在nf-κb抑制剂存在和不存在的条件下nlrp3及il-1β在mrna和蛋白水平的表达情况。使用lps刺激tim-3-tg和wt小鼠腹腔巨噬细胞,观察nlrp3和il-1β在mrna和蛋白水平的表达情况;3.tim-3调控nlrp3炎性小体的机制探讨:对复合物成分组装及活性的影响。使用tim-3-ig阻断tim-3信号通路,观察raw264.7细胞中atp释放、k+外流和ros的产生情况,并观察在k+外流和ros的产生受到抑制的情况下nlrp3炎性小体的活化情况;4.tim-3调控nlrp3炎性小体的分子机制探讨。构建tim-3分子256/263位点酪氨酸双突变体,观察这两个位点突变后tim-3对nlrp3炎性小体的表达和活化的影响情况;5.tim-3与腹膜炎患者中nlrp3炎性小体表达的关系研究。收集并检测临床腹膜炎患者血清中可溶性tim-3蛋白的表达情况,并观察人源巨噬细胞系thp-1和u937在tim-3信号通路阻断的情况下nlrp3和il-1βmrna的表达情况;6.tim-3信号对小鼠腹膜炎进程的影响。构建小鼠腹膜炎模型,观察在tim-3高表达和阻断tim-3信号通路后小鼠腹膜炎的炎症反应程度以及体内nlrp3炎症小体活化情况。结果1.和wt组相比,tim-3-tg组小鼠腹腔巨噬细胞中lps和atp诱导的nlrp3炎性小体的活化受到了抑制,表现为caspase-1活性的抑制以及il-1b分泌的显著降低。而用tim-3-ig阻断tim-3信号通路后,nlrp3炎性小体的活性被激活;2.tim-3-ig阻断tim-3信号通路后,nf-κb活性增加,nlrp3和il-1β在mrna和蛋白水平表达升高,当nf-κb受到抑制后,nlrp3和il-1β表达也受到了抑制;3.tim-3-ig阻断tim-3通路后,atp的释放、k+外流和ros的产生增加,il-1β释放增强,当k+外流和ros的产生受到抑制后,il-1β释放也受到了抑制;4.当tim-3分子256/263酪氨酸位点双突变后,tim-3对nlrp3炎性小体抑制作用减弱;5.临床腹膜炎患者血清中可溶性tim-3蛋白(stim-3)含量高于正常人。人源巨噬细胞系u937和thp-1tim-3信号通路被阻断后,NLRP3和IL-1β在mRNA水平的表达升高;6.在构建的小鼠腹膜炎中,Tim-3-TG组腹膜炎炎症反应程度明显低于WT组,Tim-3信号通路阻断组腹膜炎炎症反应程度明显高于对照组。结论1、Tim-3通过抑制NLRP3炎性小体活化的第一信号和第二信号参与NLRP3炎性小体的负性调控。2、Tim-3胞内段256/263位点的酪氨酸在其对NLRP3炎性小体的调控过程中发挥着重要的作用。3、Tim-3能够通过负性调控NLRP3炎性小体对腹膜炎发挥保护作用。
[Abstract]:In recent years, the incidence of a variety of autoimmune diseases and inflammatory diseases such as rheumatoid arthritis, gout, type I diabetes, pancreatitis, peritonitis is increasing year by year, causing a great threat to people's life and health. The study shows that there are NOD like receptors in the pathogenesis of a variety of autoimmune diseases and inflammatory diseases. The excessive activation of the inflammatory cytokines of protein 3 (NOD-like receptor, pyrin domain-containing 3, NLRP3) caused the release of the inflammatory factor of interleukin -1beta (inteleukine-1beta, IL-1 beta), thus causing severe inflammatory reactions. Therefore, the exploration of the regulatory mechanism of the NLRP3 inflammatory small body in the body and the search for its negative regulators have become scientific research. The current research suggests that the activation of NLRP3 inflammatory bodies requires two signals: preexcitation signal (first signal) and activation signal (second signal), of which the first signal is mainly responsible for the transcriptional expression of the component of NLRP3 inflammatory corpuscle complex and the effector molecules, and the second signal is the component of the NLRP3 inflammatory corpuscle. Each independent state is combined into a complex and activates the inflammatory small body of NLRP3. The NLRP3 inflammatory body in the active state can promote the maturation and release of the inflammatory factor IL-1 beta, IL-18, and cause the inflammatory response of the body and the occurrence of the.T cell immune globule mucin -3 (T-cell Ig-mucin-3, Tim-3) of the.T cells under specific conditions. The abnormal expression of.Tim-3 on T cells is related to a variety of immune related diseases such as tumor and virus infection. Therefore, it is considered to be a new generation of immunoregulation targets. In recent years, the expression and function of Tim-3 in natural immune cells are being paid more and more attention. However, Tim-3 control day However, the mechanism of immunization is still not very clear. Whether the inflammatory body, as a natural immune response component, is also regulated by Tim-3 is a very important topic. In our previous study, our laboratory found that Tim-3 can regulate the inflammatory response by inhibiting TLR4. In view of the fact that TLR4 plays an important role in the activation of NLRP3 inflammatory bodies The role of Tim-3 molecules is also involved in the regulation of the activation of NLRP3 inflammatory bodies? What is the mechanism to participate in the regulation? This will be the main content of this study. Objective to explore the role and mechanism of Tim-3 in the regulation of NLRP3 inflammatory corpuscles. Method 1.tim-3 on the activation of NLRP3 inflammatory corpuscles. Peritoneal macrophages were isolated from mice (tim-3-tg) and wild type mice (WT). The activation of NLRP3 inflammatory corpuscles in macrophages was observed by using the first signal activator LPS of NLRP3 inflammatory corpuscle and ATP of second signal activator ATP. The Tim-3 signal of RAW264.7 in rat macrophage system was blocked by Tim-3 fusion protein (tim-3-ig). Pathway, observe the activation of NLRP3 inflammatory body; the mechanism of 2.tim-3 regulating NLRP3 inflammatory corpuscle: the effect on the transcription and expression of the compound components. Using tim-3-ig to block the Tim-3 signaling pathway, observe the RAW264.7 and J774A.1 in the rat macrophage system, and the NLRP3 and IL-1 beta of NLRP3 and IL-1 beta in the mRNA and protein under the presence and absence of nf- kappa B inhibitors Level of expression. Use LPS to stimulate tim-3-tg and WT mouse peritoneal macrophages, observe the expression of NLRP3 and IL-1 beta at the level of mRNA and protein; the mechanism of 3.tim-3 regulating NLRP3 inflammatory bodies: the effect on the composition and activity of the compound composition and activity. Use tim-3-ig to block the Tim-3 signaling pathway and observe ATP release in RAW264.7 cells. Flow and ROS production, and observe the activation of NLRP3 inflammatory corpuscles in the presence of k+ Exodus and ROS production; 4.tim-3 regulating the molecular mechanism of NLRP3 inflammatory corpuscles. Construction of the Tim-3 molecule 256/263 site tyrosine double mutants to observe the expression and activation of Tim-3 to NLRP3 inflammatory corpuscles after the mutation of these two loci The relationship between the expression of 5.tim-3 and the expression of NLRP3 inflammatory corpuscle in patients with peritonitis. The expression of soluble Tim-3 protein in serum of patients with clinical peritonitis was collected and detected, and the expression of NLRP3 and IL-1 beta mRNA under the blocking of Tim-3 signaling pathway in human macrophage macrophage system was observed; 6.tim-3 signals were used in mice. The effect of peritonitis process. A mouse peritonitis model was constructed to observe the inflammatory response degree of the mouse peritonitis after Tim-3 high expression and blocking the Tim-3 signaling pathway and the activation of NLRP3 inflammatory corpuscle in the body. Results the activation of LPS and ATP induced NLRP3 inflammatory bodies induced by LPS and ATP in the peritoneal macrophages of group tim-3-tg mice was compared with that of group wt. Inhibition, the inhibition of Caspase-1 activity and the significant decrease in the secretion of IL-1B. The activity of NLRP3 inflammatory corpuscle was activated by blocking the Tim-3 signaling pathway with tim-3-ig, and nf- kappa B activity increased after 2.tim-3-ig blocked the Tim-3 signaling pathway, and NLRP3 and IL-1 beta was increased in mRNA and protein levels. When 3.tim-3-ig blocked the Tim-3 pathway, the release of ATP, the increase of k+ Exodus and ROS, the increase of IL-1 beta release, and the inhibition of IL-1 beta release when the production of k+ Exodus and ROS were inhibited; 4. when the Tim-3 molecule 256/263 tyrosine loci was double mutated, the inhibitory effect of Tim-3 on the inflammatory corpuscle was weakened; 5. clinical peritoneum The content of soluble Tim-3 protein (stim-3) in serum of inflammatory patients was higher than that of normal people. The expression of NLRP3 and IL-1 beta in mRNA level increased after the U937 and thp-1tim-3 signaling pathway was blocked in human macrophage system. 6. in the constructed mouse peritonitis, the inflammatory response of Tim-3-TG group peritonitis was significantly lower than that of the WT group, and the Tim-3 signaling pathway blocked the peritonitis. The degree of inflammatory reaction was significantly higher than that in the control group. Conclusion 1, Tim-3 participates in the negative regulation of.2 by inhibiting the first and second signals of the activation of the inflammatory corpuscle of the NLRP3, the tyrosine of the 256/263 site of the Tim-3 intracellular segment plays an important role in the regulation of the NLRP3 inflammatory corpuscle, and Tim-3 can be negatively regulated by N. LRP3 inflammatory small body plays a protective role in peritonitis.

【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R392

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