本文選題：Behcet’s病 + IL-37　； 參考：《重慶醫(yī)科大學(xué)》2016年博士論文

【摘要】：背景葡萄膜炎是一種累及葡萄膜、視網(wǎng)膜、視網(wǎng)膜血管和玻璃體的疾病,嚴(yán)重威脅患者的視力。它多發(fā)于青壯年,并可導(dǎo)致不可逆的盲目。Behcet’s病是我國(guó)致盲率最高的葡萄膜炎類型之一。它是一種多系統(tǒng)受累的自身炎癥性疾病,復(fù)發(fā)性葡萄膜炎、皮膚損害、口腔和生殖器潰瘍是此病的主要特征。Behcet’s病主要發(fā)生于“絲綢之路”沿線國(guó)家,如中國(guó)、土耳其和日本。根據(jù)以往文獻(xiàn)報(bào)道,自身免疫反應(yīng)或自身炎癥反應(yīng)在此病的發(fā)生中起著尤為重要的作用。調(diào)控異常的自身免疫反應(yīng)或自身炎癥反應(yīng)是防治Behcet’s病的有效策略。白介素37(IL-37)是最近發(fā)現(xiàn)的IL-1家族新成員,它在免疫反應(yīng)中起著重要的負(fù)性調(diào)控作用。在對(duì)動(dòng)物模型的研究中發(fā)現(xiàn),IL-37能夠顯著抑制右旋葡聚糖硫酸鈉(DSS)誘導(dǎo)的結(jié)腸炎和脂多糖誘導(dǎo)的休克模型的發(fā)生。B、T淋巴細(xì)胞衰減子(BTLA)是另一種抑制性分子,其廣泛表達(dá)于人體內(nèi)的免疫細(xì)胞表面,尤其是B細(xì)胞和T細(xì)胞表面。BTLA能夠通過與其配體單純皰疹病毒進(jìn)入介導(dǎo)子(HVEM)相互作用從而抑制過度活化的免疫細(xì)胞反應(yīng)。對(duì)BTLA基因敲除鼠的研究發(fā)現(xiàn),與野生型小鼠相比,BTLA基因敲除鼠更易發(fā)生實(shí)驗(yàn)性自身免疫性腦脊髓炎(EAE)和呼吸道炎癥。盡管IL-37和BTLA均被認(rèn)為是調(diào)節(jié)自身免疫反應(yīng)或自身炎癥反應(yīng)的抑制性分子,但它們是否參與了Behcet’s病的發(fā)生以及是否對(duì)Behcet’s病具有防治作用尚不清楚�；谝陨涎芯勘尘�,本課題主要對(duì)以下兩個(gè)問題進(jìn)行了研究:(1)IL-37和BTLA是否參與了Behcet’s病的發(fā)生和發(fā)展?(2)如果這兩個(gè)分子參與疾病的發(fā)生,它們?cè)诩膊“l(fā)生中的作用機(jī)制是什么?探討以上兩個(gè)問題,將有可能闡明Behcet’s病的發(fā)生機(jī)制,并提供防治Behcet’s病的新靶點(diǎn)。第一部分IL-37在Behcet’s病發(fā)病機(jī)制中的作用研究目的:IL-37在固有免疫反應(yīng)中起著重要的調(diào)節(jié)作用。在本部分研究中,我們探討IL-37在Behcet’s病患者中的表達(dá)水平,以及它在此病的發(fā)病過程中可能的作用機(jī)制。方法:1.利用熒光定量PCR(RT-PCR)和流式細(xì)胞術(shù)檢測(cè)IL-37在Behcet’s病患者和正常人外周血單個(gè)核細(xì)胞(PBMCs)中的表達(dá)。2.利用酶聯(lián)免疫吸附技術(shù)探討重組人IL-37對(duì)單核細(xì)胞誘導(dǎo)的樹突狀細(xì)胞(DCs)分泌細(xì)胞因子的影響。3.利用流式細(xì)胞術(shù)探討IL-37對(duì)DC表面標(biāo)志的表達(dá)、活性氧的水平和絲裂原活化蛋白激酶(MAPK)信號(hào)通路的磷酸化的作用。4.利用ELISAs和流式細(xì)胞術(shù)探討IL-37預(yù)處理的DC對(duì)Th17細(xì)胞和Th1細(xì)胞分化的作用。結(jié)果:1.與正常對(duì)照相比,活動(dòng)期Behcet’s病患者體內(nèi)IL-37的m RNA水平和蛋白水平均有顯著下降。2.IL-37能夠顯著抑制Behcet’s病患者和正常人體內(nèi)DC分泌促炎性細(xì)胞因子IL-6、IL-1β和TNF-α,同時(shí)促進(jìn)調(diào)節(jié)性細(xì)胞因子IL-27的產(chǎn)生。3.IL-37對(duì)DC細(xì)胞內(nèi)活性氧的產(chǎn)生以及MAPK的三個(gè)信號(hào)通路ERK1/2、JNK和p38 MAPK的磷酸化均有明顯的抑制作用。4.IL-37預(yù)處理的DC能夠顯著抑制Th17細(xì)胞和Th1細(xì)胞分化,同時(shí)抑制CD4+T細(xì)胞分泌IL-17和IFN-γ。5.IL-37對(duì)DC細(xì)胞表面的五種表面標(biāo)志CD83、CD86、CD80、CD40和HLA-DR沒有明顯作用,對(duì)DC分泌IL-10也沒有影響。結(jié)論:我們的研究發(fā)現(xiàn)在活動(dòng)性Behcet’s病患者體內(nèi)IL-37的表達(dá)顯著降低,可能導(dǎo)致其無(wú)法有效發(fā)揮對(duì)DC分泌促炎性細(xì)胞因子和產(chǎn)生活性氧的抑制作用以及對(duì)Th17細(xì)胞和Th1細(xì)胞分化的調(diào)節(jié)作用,從而參與到Behcet’s病的發(fā)生。第二部分BTLA在Behcet’s病發(fā)病機(jī)制中的作用研究目的:Behcet’s病是一種嚴(yán)重?fù)p害視力的疾病。已有研究報(bào)道Th17細(xì)胞和Th1細(xì)胞的過度激活與此病的發(fā)生密切相關(guān)。BTLA已被證實(shí)在免疫反應(yīng)中起著重要的調(diào)節(jié)作用。本研究主要探討B(tài)TLA的活化是否能夠調(diào)節(jié)Behcet’s病中異常的免疫反應(yīng),以及它在此病的發(fā)病過程中的作用及其可能機(jī)制。方法:1.抽取活動(dòng)期Behcet’s病患者和健康對(duì)照的外周血,分離PBMCs。利用磁珠分選CD4+T細(xì)胞或CD14+單核細(xì)胞,利用流式細(xì)胞術(shù)將CD4+T細(xì)胞分選為BTLAhi和BTLAlo兩群細(xì)胞。2.利用RT-PCR和流式細(xì)胞術(shù)檢測(cè)Behcet’s病患者和健康對(duì)照體內(nèi)PBMCs表面BTLA的表達(dá)水平;利用流式細(xì)胞術(shù)檢測(cè)Behcet’s病患者和健康對(duì)照體內(nèi)PBMCs不同亞群表面BTLA的表達(dá)情況。3.利用流式細(xì)胞術(shù)檢測(cè)患者和健康對(duì)照體內(nèi)Th17細(xì)胞和Th1細(xì)胞比例,以及產(chǎn)生IL-17和IFN-γ的BTLAhiCD4+T細(xì)胞和BTLAloCD4+T細(xì)胞比例。4.利用BTLA特異性激動(dòng)劑活化BTLA,并評(píng)價(jià)其對(duì)患者和健康對(duì)照體內(nèi)IL-17、IFN-γ和IL-22分泌的影響和對(duì)Th17細(xì)胞、Th1細(xì)胞比例的影響。5.利用流式細(xì)胞術(shù)和ELISAs檢測(cè)BTLA活化對(duì)DC誘導(dǎo)的Th17細(xì)胞和Th1細(xì)胞免疫反應(yīng)的影響。6.利用流式細(xì)胞術(shù)和ELISAs評(píng)價(jià)BTLA活化對(duì)DC表面標(biāo)志物的影響及對(duì)DC分泌Th17細(xì)胞、Th1細(xì)胞活化相關(guān)細(xì)胞因子的影響。結(jié)果:1.活動(dòng)期Behcet’s病患者體內(nèi)BTLA m RNA及蛋白水平顯著低于健康對(duì)照;患者體內(nèi)BTLA在CD4+T細(xì)胞表面的表達(dá)也明顯降低。2.活動(dòng)期Behcet’s病患者體內(nèi)Th17細(xì)胞和Th1細(xì)胞比例均顯著高于健康對(duì)照,活動(dòng)期Behcet’s病患者Th17細(xì)胞和Th1細(xì)胞免疫反應(yīng)過度活化。3.經(jīng)anti-CD3和anti-CD28預(yù)處理后,患者體內(nèi)的BTLAloIL-17+細(xì)胞比例顯著高于正常人,同時(shí)BTLAloCD4+T細(xì)胞表達(dá)更多IL-17和IFN-γ。4.BTLA特異性激動(dòng)劑活化BTLA后,患者和健康對(duì)照的CD4+T細(xì)胞分泌的IL-17、IL-22和IFN-γ均顯著降低,同時(shí)患者和健康對(duì)照體內(nèi)的Th17細(xì)胞和Th1細(xì)胞比例也明顯下降。5.活化BTLA明顯抑制DC細(xì)胞誘導(dǎo)的Th17細(xì)胞和Th1細(xì)胞活化,并抑制CD4+T細(xì)胞分泌IL-17和IFN-γ。6.活化BTLA顯著抑制DC表面與T細(xì)胞活化相關(guān)的分子CD40的表達(dá),同時(shí)抑制DC分泌Th17細(xì)胞和Th1細(xì)胞活化相關(guān)細(xì)胞因子IL-1β、IL-6、IL-23和IL-12p70。結(jié)論:我們的研究發(fā)現(xiàn),活動(dòng)性Behcet’s病患者體內(nèi)BTLA表達(dá)降低與Th17細(xì)胞和Th1細(xì)胞的過度活化密切相關(guān)。BTLA的表達(dá)降低可能影響其對(duì)Th17細(xì)胞和Th1細(xì)胞免疫反應(yīng)以及DC功能的調(diào)節(jié)作用,從而參與到疾病的發(fā)生和復(fù)發(fā)。利用BTLA特異性激動(dòng)劑活化BTLA可能成為防治Behcet’s病的一個(gè)新策略。
[Abstract]:Background uveitis is a disease that involves the grape membrane, the retina, the retinal vessels and the vitreous body, which seriously threatens the patient's eyesight. It often occurs in young and young adults, and causes irreversible blind.Behcet 's disease to be one of the type of uveitis with the highest rate of blindness in China. It is a kind of multiple systemic inflammatory disease with relapse. Uveitis, skin damage, oral and genital ulcers are the main features of the disease,.Behcet 's disease mainly occurs along the "Silk Road" countries, such as China, Turkey and Japan. According to the previous literature, autoimmune reactions or self inflammatory reactions play a particularly important role in the occurrence of this disease. Immune response or self inflammatory response is an effective strategy for the prevention and treatment of Behcet 's disease. IL-37 (IL-37) is a recently discovered new member of the IL-1 family. It plays an important negative regulatory role in the immune response. In the study of animal models, IL-37 can significantly inhibit the colitis and lipoid induced by dextran sodium sulfate (DSS). The shock model of glucose induced.B, T lymphocyte attenuator (BTLA) is another inhibitory molecule, which is widely expressed in the surface of the immune cells in the human body, especially on the surface of B and T cells, which can inhibit the overactivated immune response by interacting with the ligand herpes simplex virus into the mediator (HVEM). The study of BTLA gene knockout mice found that BTLA gene knockout mice were more likely to have experimental autoimmune encephalomyelitis (EAE) and respiratory inflammation compared with wild type mice. Although both IL-37 and BTLA were considered to be an inhibitory part of the autoimmune reaction or self inflammatory response, they were involved in the occurrence of Behcet 's disease. And whether the effect of Behcet 's disease is not clear. Based on the above research background, this topic is mainly to study the following two questions: (1) does IL-37 and BTLA participate in the occurrence and development of Behcet' s disease? (2) what is the mechanism of their role in the disease if these two molecules are involved in the disease? The above two questions will be possible to elucidate the pathogenesis of Behcet 's disease and provide a new target for the prevention and treatment of Behcet' s disease. Part 1 Research on the role of IL-37 in the pathogenesis of Behcet 's disease: IL-37 plays an important regulatory role in the inherent immune response. In this part, we explore IL-37 in Behcet' s' patients. Expression level and its possible mechanism of action in the pathogenesis of this disease. Methods: 1. the expression of IL-37 in Behcet 's disease patients and normal human peripheral blood mononuclear cells (PBMCs) was detected by fluorescence quantitative PCR (RT-PCR) and flow cytometry. The enzyme linked immunosorbent assay (ELISA) was used to explore the tree induced by recombinant human IL-37 to mononuclear cells. The effect of DCs secreting cytokine.3. using flow cytometry to investigate the expression of IL-37 on the surface markers of DC, the level of reactive oxygen species and the phosphorylation of mitogen activated protein kinase (MAPK) signaling pathway,.4. using ELISAs and flow cytometry to explore the effect of DC on Th17 cells and Th1 cells in IL-37 pretreated. Results: 1. compared with the normal control, the m RNA level and protein level of IL-37 in patients with active Behcet 's's disease decreased significantly and.2.IL-37 significantly inhibited the DC secreting proinflammatory cytokines IL-6, IL-1 beta and TNF- alpha in Behcet' s patients and normal individuals, and promoted the intracellular activity of regulatory fine cell factor. The production of oxygen and the three signaling pathways of MAPK, ERK1/2, JNK and p38 MAPK have obvious inhibitory effects on.4.IL-37 pretreated DC, which can significantly inhibit the differentiation of Th17 cells and Th1 cells, and inhibit the IL-17 and IFN- gamma radiation on the five surface markers of the surface of the cells. Explicit action has no effect on the secretion of IL-10 by DC. Conclusion: our study found that the expression of IL-37 in active Behcet 's patients was significantly reduced, which could lead to the inhibition of the inhibitory effect of DC secreting cytokines and the production of living oxygen, as well as the regulation of the differentiation of Th17 fine cells and Th1 cells. The occurrence of Behcet 's disease. Second part of the role of BTLA in the pathogenesis of Behcet' s disease: Behcet 's disease is a serious impairment of visual acuity. It has been reported that the excessive activation of Th17 cells and Th1 cells is closely related to the occurrence of this disease..BTLA has been proved to play an important regulatory role in the immune response. The main discussion is whether activation of BTLA can regulate the abnormal immune response in Behcet 's disease, and its role in the pathogenesis of this disease and its possible mechanism. Methods: 1. extract the peripheral blood of patients with Behcet' disease and healthy controls at active stage, separate PBMCs. using magnetic beads to separate CD4+T cells or CD14+ mononuclear cells, and use flow cells. CD4+T cells were selected as BTLAhi and BTLAlo two group cells.2. using RT-PCR and flow cytometry to detect the expression level of BTLA on the PBMCs surface of Behcet 's disease patients and healthy controls. Flow cytometry was used to detect the expression of PBMCs different subgroups in Behcet' s' patients and healthy controls. The proportion of Th17 and Th1 cells in patients and healthy controls, as well as the proportion of BTLAhiCD4+T and BTLAloCD4+T cells producing IL-17 and IFN- gamma,.4. using BTLA specific agonists to activate BTLA, and to evaluate the effects on IL-17, IFN- gamma and IL-22 secretion in patients and healthy controls. The effects of BTLA activation on DC induced Th17 and Th1 cell immune responses by flow cytometry and ELISAs.6. use flow cytometry and ELISAs to evaluate the effect of BTLA activation on DC surface markers and the effect on DC secreted Th17 cells and Th1 cell activation related cytokines. Results: 1. The expression of protein and protein was significantly lower than that of healthy controls, and the expression of BTLA on the surface of CD4+T cells in the patients was also significantly lower than that of Th17 cells and Th1 cells in the Behcet 's disease patients. The activity of Th17 cells and Th1 cells in the active Behcet' s disease patients' Th17 cells and Th1 cells After that, the proportion of BTLAloIL-17+ cells in the patients was significantly higher than that of the normal human, while the BTLAloCD4+T cells expressed more IL-17 and IFN- gamma.4.BTLA specific agonists activated BTLA, and the IL-17, IL-22 and IFN- gamma secreted by the healthy control CD4+T cells decreased significantly, while the patients and the Th17 cells and Th1 cells in the healthy control were compared to those of the healthy control. The.5. activation BTLA significantly inhibited the activation of Th17 and Th1 cells induced by DC cells, and the inhibition of the secretion of IL-17 and IFN- gamma.6. activated BTLA in CD4+T cells significantly inhibited the expression of the DC surface associated with the activation of T cells. 70. conclusion: our study found that the decrease of BTLA expression in patients with active Behcet 's's disease is closely related to the overactivation of Th17 cells and Th1 cells. The decrease of.BTLA expression may affect the regulation of the immune response and DC function of Th17 cells and Th1 cells, and thus participate in the occurrence and recurrence of the disease. BTLA specific excitation is used. Activating BTLA may become a new strategy for the prevention and treatment of Behcet 's disease.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R773.9

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