本文選題：Behcet病 + Vogt-小柳原田綜合征�。� 參考：《重慶醫(yī)科大學(xué)》2013年博士論文

【摘要】：背景 葡萄膜炎是指累及葡萄膜、視網(wǎng)膜、視網(wǎng)膜血管和玻璃體的一大類炎癥性疾病的總稱。根據(jù)葡萄膜炎病因及臨床表現(xiàn)不同，可將其分為100余種。葡萄膜炎多發(fā)于青壯年，表現(xiàn)為慢性或反復(fù)發(fā)作的眼部炎癥，且其造成的眼組織損害是不可逆的，可導(dǎo)致不可逆性盲目。Behcet病和Vogt-小柳原田綜合征是我國各種葡萄膜炎中最為常見也是致盲率最高的葡萄膜炎類型。目前認(rèn)為，Behcet病為一種自身炎癥性疾病，而Vogt-小柳原田綜合征為一種自身免疫性疾病。兩者的病因目前還不清楚，但感染和免疫反應(yīng)被認(rèn)為均參與了兩種疾病的發(fā)生。模式識(shí)別受體（pattern-recognition receptors，PRR）是固有免疫的重要組成部分，在機(jī)體抵抗外來微生物感染中發(fā)揮了重要的作用。模式識(shí)別受體在接觸到外來微生物后可以將其轉(zhuǎn)化為適應(yīng)性免疫，激活免疫細(xì)胞，促進(jìn)炎癥因子釋放，進(jìn)而引發(fā)炎癥反應(yīng)消除外來微生物。但是，模式識(shí)別受體表達(dá)及功能的異�？梢詫�(dǎo)致免疫系統(tǒng)紊亂，從而導(dǎo)致疾病的發(fā)生。 本研究正是基于上述背景，進(jìn)行了以下兩大方面的實(shí)驗(yàn)：1、探討模式識(shí)別受體在Behcet病和Vogt-小柳原田綜合征患者中的表達(dá)，以期尋找到差異表達(dá)的模式識(shí)別受體。2、進(jìn)一步探討差異表達(dá)的模式識(shí)別受體在Behcet病和Vogt-小柳原田綜合征發(fā)病中所起的作用。通過上述研究，以期能對(duì)以上兩種疾病的發(fā)病機(jī)制有一個(gè)深入的了解，，同時(shí)為其預(yù)防和治療尋找到新的靶點(diǎn)和突破口。 第一部分模式識(shí)別受體在Behcet病發(fā)病中作用的研究 目的： 探討模式識(shí)別受體在Behcet病發(fā)病中的作用。 方法： 分別抽取活動(dòng)期Behcet病患者、靜止期的Behcet病患者、急性前葡萄膜炎患者（acute anterior uveitis, AAU）和正常人外周靜脈血，分離單核細(xì)胞并誘導(dǎo)為外周血單核細(xì)胞誘導(dǎo)的巨噬細(xì)胞（monocyte-derived macrophages，MDMs）。使用熒光定量PCR技術(shù)及流式細(xì)胞技術(shù)檢測TLR2/4的表達(dá)；使用ELISA方法檢測IL-1β表達(dá)水平；使用流式細(xì)胞技術(shù)檢測線粒體受損及活性氧簇（reactive oxygenspecies，ROS）產(chǎn)生水平；使用基因沉默技術(shù)使NLRP3低表達(dá)；使用流式細(xì)胞技術(shù)評(píng)價(jià)MAPK信號(hào)通路磷酸化水平。 結(jié)果： 1.活動(dòng)期Behcet病患者M(jìn)DMs細(xì)胞TLR2和TLR4表達(dá)明顯高于靜止期Behcet病患者、AAU患者和正常人。 2.活動(dòng)期Behcet病患者M(jìn)DMs產(chǎn)生IL-1β水平明顯高于靜止期Behcet病患者、AAU患者和正常人。PGN和LPS刺激MDMs后，可以明顯升高各組IL-1β產(chǎn)生水平。但是，活動(dòng)期Behcet病患者IL-1β升高水平明顯高于其它各實(shí)驗(yàn)組，其它各實(shí)驗(yàn)組之間無明顯差異。 3.活動(dòng)期Behcet病患者體內(nèi)MDMs細(xì)胞線粒體受損及ROS產(chǎn)生水平與靜止期Behcet病患者、AAU患者和正常人相比明顯升高；PGN和LPS可以使各組MDMs細(xì)胞線粒體受損及ROS產(chǎn)生水平明顯增高，并且活動(dòng)期Behcet病患者升高水平明顯高于其它組；anti-TLR2/anti-TLR4可以明顯降低PGN/LPS對(duì)MDMs細(xì)胞線粒體損傷和ROS產(chǎn)生作用；rotenone可以明顯升高M(jìn)DMs細(xì)胞ROS產(chǎn)生水平及IL-1β分泌水平，而DPI可以明顯降低MDMs細(xì)胞ROS產(chǎn)生水平及IL-1β分泌水平。 4.基因沉默炎癥小體NLRP3后，各組IL-1β產(chǎn)生顯著降低。 5. ROS通過激活p38和ERK1/2，進(jìn)而激活炎癥小體NLRP3，從而使IL-1β產(chǎn)生增多。 結(jié)論： 我們的結(jié)果顯示：TLR2/4在活動(dòng)期Behcet病患者M(jìn)DMs細(xì)胞中表達(dá)明顯升高。TLR2/4與其配體PGN/LPS相互作用可以使線粒體產(chǎn)生ROS增多，進(jìn)而激活炎癥小體NLRP3，從而使IL-1β產(chǎn)生增多引起炎癥反應(yīng)。 第二部分模式識(shí)別受體在Vogt-小柳原田綜合征發(fā)病中作用的研究 目的： 探討模式識(shí)別受體在Vogt-小柳原田綜合征發(fā)病中所起的作用。 方法： 分別抽取活動(dòng)期Vogt-小柳原田綜合征患者、靜止期Vogt-小柳原田綜合征患者、AAU患者和正常人外周靜脈血，分離單核細(xì)胞并誘導(dǎo)為巨噬細(xì)胞。使用熒光定量PCR技術(shù)及流式細(xì)胞技術(shù)檢測TLR2/3/4的表達(dá)；使用ELISA方法檢測IL-1β表達(dá)水平；使用流式細(xì)胞技術(shù)檢測線粒體受損及活性氧簇（ROS）產(chǎn)生水平；使用基因沉默技術(shù)使NLRP3低表達(dá)；使用流式細(xì)胞技術(shù)評(píng)價(jià)MAPK信號(hào)通路磷酸化水平。 結(jié)果： 1.活動(dòng)期Vogt-小柳原田綜合征患者M(jìn)DMs細(xì)胞TLR3和TLR4表達(dá)明顯高于靜止期Vogt-小柳原田綜合征患者、AAU患者和正常人;TLR2表達(dá)在各組之間無明顯差異。 2.活動(dòng)期Vogt-小柳原田綜合征患者M(jìn)DMs產(chǎn)生IL-1β水平明顯高于靜止期Vogt-小柳原田綜合征患者、AAU患者和正常人。Poly I:C和LPS刺激MDMs后，可以明顯升高各組IL-1β產(chǎn)生水平。但是，活動(dòng)期Vogt-小柳原田綜合征患者IL-1β升高水平明顯高于其它各實(shí)驗(yàn)組，其它各實(shí)驗(yàn)組之間無明顯差異。 3.活動(dòng)期Vogt-小柳原田綜合征患者體內(nèi)MDMs細(xì)胞線粒體受損及ROS產(chǎn)生水平與靜止期Vogt-小柳原田綜合征患者、AAU患者和正常人相比明顯升高；poly I:C和LPS可以使各組MDMs細(xì)胞線粒體受損及ROS產(chǎn)生水平明顯增高，并且活動(dòng)期Vogt-小柳原田綜合征患者升高水平明顯高于其它組；anti-TLR3/anti-TLR4可以明顯降低poly I:C/LPS對(duì)MDMs細(xì)胞線粒體損傷和ROS產(chǎn)生的作用；rotenone可以明顯升高M(jìn)DMs細(xì)胞ROS產(chǎn)生水平及IL-1β產(chǎn)生水平，而DPI可以明顯降低MDMs細(xì)胞ROS產(chǎn)生水平及IL-1β產(chǎn)生水平。 4.基因沉默炎癥小體NLRP3后，各組IL-1β產(chǎn)生水平顯著降低。 5. ROS通過激活p38和ERK1/2信號(hào)通路進(jìn)而激活炎癥小體NLRP3，從使IL-1β產(chǎn)生增多。 結(jié)論： 我們的結(jié)果顯示：TLR3/4在活動(dòng)期Vogt-小柳原田綜合征患者M(jìn)DMs細(xì)胞中表達(dá)明顯升高。TLR3/4與其配體poly I:C/LPS相互作用可以通過使線粒體產(chǎn)生ROS增多，激活炎癥小體NLRP3，進(jìn)而使IL-1β產(chǎn)生增多引起炎癥反應(yīng)。
[Abstract]:background
Uveitis is the general name of a large type of inflammatory disease involving the Vineum, retina, retinal vessels and vitreous body. According to the etiology and clinical manifestations of uveitis, it can be divided into more than 100 species. Uveitis is more likely to occur in young and young adults, characterized by chronic or recurrent ocular inflammation, and its ocular tissue damage is irreversible. It can lead to irreversible blindness.Behcet's disease and Vogt- small Liu Yuantian syndrome, the most common type of uveitis in various uveitis and the highest blinding rate in our country. At present, Behcet's disease is a self inflammatory disease, and Vogt- small Liu Yuantian syndrome is a self immune disease. The cause of the two is not yet clear. Chu, but infection and immune response are considered to be involved in the occurrence of two diseases. Pattern-recognition receptors (PRR) is an important part of the inherent immunity and plays an important role in the body's resistance to external microbial infections. Sexual immunity, activating immune cells, promoting the release of inflammatory factors and causing inflammatory reactions to eliminate foreign microorganisms. However, abnormal expression and function of pattern recognition receptors can lead to the disorder of the immune system, which leads to the occurrence of disease.
This study is based on the above background and carries out the following two major experiments: 1, to explore the expression of pattern recognition receptors in patients with Behcet's disease and Vogt- small Liu Yuantian's syndrome, in order to find the differentially expressed pattern recognition receptor.2 and further explore the differential expression pattern recognition receptors in Behcet's disease and Vogt- small Liu Yuantian syndrome The role of the disease, through the above study, is expected to have an in-depth understanding of the pathogenesis of the above two diseases, and to find new targets and breakthroughs for its prevention and treatment.
Part I the role of pattern recognition receptors in the pathogenesis of Behcet's disease
Objective:
Objective to investigate the role of pattern recognition receptors in the pathogenesis of Behcet disease.
Method:
The patients with active Behcet disease, Behcet disease at rest, acute anterior uveitis (acute anterior uveitis, AAU) and normal human peripheral venous blood were isolated and isolated from mononuclear cells (monocyte-derived macrophages, MDMs) induced by peripheral blood mononuclear cells (monocyte-derived macrophages, MDMs). Fluorescence quantitative PCR technique and flow cytometry were used. The expression of TLR2/4 was detected by technique; the expression level of IL-1 beta was detected by ELISA method; the level of mitochondrial damage and reactive oxygen cluster (reactive OXYGENSPECIES, ROS) production was detected by flow cytometry; the expression of NLRP3 was reduced by gene silencing, and the level of phosphorylation of MAPK signaling pathway was evaluated by flow cytometry.
Result錛

本文編號(hào)：2053632