本文選題：Tim-3 + NLRP3炎性小體　； 參考：《河南大學(xué)》2016年碩士論文

【摘要】：背景近年來,多種自身免疫性疾病及炎癥性疾病如類風(fēng)濕性關(guān)節(jié)炎、痛風(fēng)、Ⅰ型糖尿病、胰腺炎、腹膜炎等發(fā)病率逐年升高,對(duì)人們生命健康造成了極大的威脅。研究表明,在多種自身免疫性疾病以及炎癥性疾病的發(fā)病過程中存在NOD樣受體蛋白3(NOD-like receptor,pyrin domain-containing 3,NLRP3)炎癥小體的過度活化引起的炎癥因子白細(xì)胞介素-1beta(inteleukine-1beta,IL-1β)大量釋放,進(jìn)而引起劇烈的炎癥反應(yīng)。所以,探索機(jī)體中NLRP3炎癥小體的調(diào)控機(jī)制及尋找其負(fù)性調(diào)控分子成為了科研工作者迫切要解決的課題。目前研究認(rèn)為,NLRP3炎癥小體的活化需要兩個(gè)信號(hào):預(yù)激信號(hào)(第一信號(hào))和激活信號(hào)(第二信號(hào)),其中第一信號(hào)主要負(fù)責(zé)NLRP3炎性小體復(fù)合體組分和效應(yīng)分子的轉(zhuǎn)錄表達(dá),第二信號(hào)則是將NLRP3炎性小體各組分由各自獨(dú)立狀態(tài)組合成復(fù)合體,同時(shí)激活NLRP3炎性小體,處于活化狀態(tài)的NLRP3炎性小體可以促進(jìn)炎癥因子IL-1β、IL-18的成熟與釋放,從而引起機(jī)體的炎癥反應(yīng)和特定條件下疾病的發(fā)生。T細(xì)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檢查點(diǎn)分子。Tim-3在T細(xì)胞上的異常表達(dá)與多種免疫相關(guān)性疾病如腫瘤、病毒感染等相關(guān),因此被認(rèn)為是新一代的免疫調(diào)控靶點(diǎn)。近年來,Tim-3在天然免疫細(xì)胞上的表達(dá)及功能越來越受到人們的重視,然而Tim-3調(diào)控天然免疫的機(jī)制尚不十分清楚。作為天然免疫效應(yīng)成分的炎癥小體是否也受到Tim-3調(diào)控是非常值得探討的課題。我們實(shí)驗(yàn)室在前期研究中發(fā)現(xiàn)Tim-3能夠通過抑制TLR4參與調(diào)控炎癥反應(yīng)。鑒于TLR4在NLRP3炎性小體的活化過程中也發(fā)揮著重要的作用,那么Tim-3分子是否也參與調(diào)控NLRP3炎癥小體的活化?其參與調(diào)控的機(jī)制是什么?這將是本課題研究的主要內(nèi)容。目的探討Tim-3對(duì)NLRP3炎癥小體的調(diào)控作用及其機(jī)制。方法1.tim-3對(duì)nlrp3炎性小體活化作用的探討。從tim-3高表達(dá)的轉(zhuǎn)基因小鼠(tim-3-tg)和野生型小鼠(wt)中分離出腹腔巨噬細(xì)胞,使用nlrp3炎性小體活化的第一信號(hào)激活劑lps和第二信號(hào)激活劑atp進(jìn)行刺激,觀察巨噬細(xì)胞中nlrp3炎性小體的活化情況。使用tim-3融合蛋白(tim-3-ig)阻斷鼠源巨噬細(xì)胞系raw264.7中tim-3信號(hào)通路,觀察nlrp3炎性小體的活化情況;2.tim-3調(diào)控nlrp3炎性小體的機(jī)制探討:對(duì)復(fù)合物成分轉(zhuǎn)錄及表達(dá)的影響。使用tim-3-ig阻斷tim-3信號(hào)通路,觀察鼠源巨噬細(xì)胞系raw264.7以及j774a.1在nf-κb抑制劑存在和不存在的條件下nlrp3及il-1β在mrna和蛋白水平的表達(dá)情況。使用lps刺激tim-3-tg和wt小鼠腹腔巨噬細(xì)胞,觀察nlrp3和il-1β在mrna和蛋白水平的表達(dá)情況;3.tim-3調(diào)控nlrp3炎性小體的機(jī)制探討:對(duì)復(fù)合物成分組裝及活性的影響。使用tim-3-ig阻斷tim-3信號(hào)通路,觀察raw264.7細(xì)胞中atp釋放、k+外流和ros的產(chǎn)生情況,并觀察在k+外流和ros的產(chǎn)生受到抑制的情況下nlrp3炎性小體的活化情況;4.tim-3調(diào)控nlrp3炎性小體的分子機(jī)制探討。構(gòu)建tim-3分子256/263位點(diǎn)酪氨酸雙突變體,觀察這兩個(gè)位點(diǎn)突變后tim-3對(duì)nlrp3炎性小體的表達(dá)和活化的影響情況;5.tim-3與腹膜炎患者中nlrp3炎性小體表達(dá)的關(guān)系研究。收集并檢測臨床腹膜炎患者血清中可溶性tim-3蛋白的表達(dá)情況,并觀察人源巨噬細(xì)胞系thp-1和u937在tim-3信號(hào)通路阻斷的情況下nlrp3和il-1βmrna的表達(dá)情況;6.tim-3信號(hào)對(duì)小鼠腹膜炎進(jìn)程的影響。構(gòu)建小鼠腹膜炎模型,觀察在tim-3高表達(dá)和阻斷tim-3信號(hào)通路后小鼠腹膜炎的炎癥反應(yīng)程度以及體內(nèi)nlrp3炎癥小體活化情況。結(jié)果1.和wt組相比,tim-3-tg組小鼠腹腔巨噬細(xì)胞中l(wèi)ps和atp誘導(dǎo)的nlrp3炎性小體的活化受到了抑制,表現(xiàn)為caspase-1活性的抑制以及il-1b分泌的顯著降低。而用tim-3-ig阻斷tim-3信號(hào)通路后,nlrp3炎性小體的活性被激活;2.tim-3-ig阻斷tim-3信號(hào)通路后,nf-κb活性增加,nlrp3和il-1β在mrna和蛋白水平表達(dá)升高,當(dāng)nf-κb受到抑制后,nlrp3和il-1β表達(dá)也受到了抑制;3.tim-3-ig阻斷tim-3通路后,atp的釋放、k+外流和ros的產(chǎn)生增加,il-1β釋放增強(qiáng),當(dāng)k+外流和ros的產(chǎn)生受到抑制后,il-1β釋放也受到了抑制;4.當(dāng)tim-3分子256/263酪氨酸位點(diǎn)雙突變后,tim-3對(duì)nlrp3炎性小體抑制作用減弱;5.臨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。人源巨噬細(xì)胞系u937和thp-1tim-3信號(hào)通路被阻斷后,NLRP3和IL-1β在mRNA水平的表達(dá)升高;6.在構(gòu)建的小鼠腹膜炎中,Tim-3-TG組腹膜炎炎癥反應(yīng)程度明顯低于WT組,Tim-3信號(hào)通路阻斷組腹膜炎炎癥反應(yīng)程度明顯高于對(duì)照組。結(jié)論1、Tim-3通過抑制NLRP3炎性小體活化的第一信號(hào)和第二信號(hào)參與NLRP3炎性小體的負(fù)性調(diào)控。2、Tim-3胞內(nèi)段256/263位點(diǎn)的酪氨酸在其對(duì)NLRP3炎性小體的調(diào)控過程中發(fā)揮著重要的作用。3、Tim-3能夠通過負(fù)性調(diào)控NLRP3炎性小體對(duì)腹膜炎發(fā)揮保護(hù)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.

【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R392

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6 周航;小檗堿通過抑制NLRP3炎癥小體活化改善小鼠胰島素抵抗[D];南京大學(xué);2014年

7 楊加偉;糖皮質(zhì)激素調(diào)控NLRP3炎性體表達(dá)在ALI/ARDS中的作用和機(jī)制[D];蘇州大學(xué);2016年

8 尹聰聰;NLRP3炎癥小體信號(hào)通路基因多態(tài)性與骨髓增生異常綜合征易感性的關(guān)聯(lián)研究[D];山東大學(xué);2016年

9 許鍵;NLRP3炎癥小體與心房顫動(dòng)的相關(guān)性研究[D];廣西醫(yī)科大學(xué);2016年

10 張維康;NLRP3炎癥復(fù)合體在呼吸機(jī)相關(guān)性肺損傷中的作用機(jī)制研究[D];廣西醫(yī)科大學(xué);2016年

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