白介素37和B、T淋巴细胞衰减子在Behcet’s病发病机制中的作用研究

发布时间：2018-04-28 02:17

本文选题：Behcet’s病 + IL-37　；参考：《重庆医科大学》2016年博士论文

【摘要】：背景葡萄膜炎是一种累及葡萄膜、视网膜、视网膜血管和玻璃体的疾病,严重威胁患者的视力。它多发于青壮年,并可导致不可逆的盲目。Behcet’s病是我国致盲率最高的葡萄膜炎类型之一。它是一种多系统受累的自身炎症性疾病,复发性葡萄膜炎、皮肤损害、口腔和生殖器溃疡是此病的主要特征。Behcet’s病主要发生于“丝绸之路”沿线国家,如中国、土耳其和日本。根据以往文献报道,自身免疫反应或自身炎症反应在此病的发生中起着尤为重要的作用。调控异常的自身免疫反应或自身炎症反应是防治Behcet’s病的有效策略。白介素37(IL-37)是最近发现的IL-1家族新成员,它在免疫反应中起着重要的负性调控作用。在对动物模型的研究中发现,IL-37能够显著抑制右旋葡聚糖硫酸钠(DSS)诱导的结肠炎和脂多糖诱导的休克模型的发生。B、T淋巴细胞衰减子(BTLA)是另一种抑制性分子,其广泛表达于人体内的免疫细胞表面,尤其是B细胞和T细胞表面。BTLA能够通过与其配体单纯疱疹病毒进入介导子(HVEM)相互作用从而抑制过度活化的免疫细胞反应。对BTLA基因敲除鼠的研究发现,与野生型小鼠相比,BTLA基因敲除鼠更易发生实验性自身免疫性脑脊髓炎(EAE)和呼吸道炎症。尽管IL-37和BTLA均被认为是调节自身免疫反应或自身炎症反应的抑制性分子,但它们是否参与了Behcet’s病的发生以及是否对Behcet’s病具有防治作用尚不清楚。基于以上研究背景,本课题主要对以下两个问题进行了研究:(1)IL-37和BTLA是否参与了Behcet’s病的发生和发展?(2)如果这两个分子参与疾病的发生,它们在疾病发生中的作用机制是什么?探讨以上两个问题,将有可能阐明Behcet’s病的发生机制,并提供防治Behcet’s病的新靶点。第一部分IL-37在Behcet’s病发病机制中的作用研究目的:IL-37在固有免疫反应中起着重要的调节作用。在本部分研究中,我们探讨IL-37在Behcet’s病患者中的表达水平,以及它在此病的发病过程中可能的作用机制。方法:1.利用荧光定量PCR(RT-PCR)和流式细胞术检测IL-37在Behcet’s病患者和正常人外周血单个核细胞(PBMCs)中的表达。2.利用酶联免疫吸附技术探讨重组人IL-37对单核细胞诱导的树突状细胞(DCs)分泌细胞因子的影响。3.利用流式细胞术探讨IL-37对DC表面标志的表达、活性氧的水平和丝裂原活化蛋白激酶(MAPK)信号通路的磷酸化的作用。4.利用ELISAs和流式细胞术探讨IL-37预处理的DC对Th17细胞和Th1细胞分化的作用。结果:1.与正常对照相比,活动期Behcet’s病患者体内IL-37的m RNA水平和蛋白水平均有显著下降。2.IL-37能够显著抑制Behcet’s病患者和正常人体内DC分泌促炎性细胞因子IL-6、IL-1β和TNF-α,同时促进调节性细胞因子IL-27的产生。3.IL-37对DC细胞内活性氧的产生以及MAPK的三个信号通路ERK1/2、JNK和p38 MAPK的磷酸化均有明显的抑制作用。4.IL-37预处理的DC能够显著抑制Th17细胞和Th1细胞分化,同时抑制CD4+T细胞分泌IL-17和IFN-γ。5.IL-37对DC细胞表面的五种表面标志CD83、CD86、CD80、CD40和HLA-DR没有明显作用,对DC分泌IL-10也没有影响。结论:我们的研究发现在活动性Behcet’s病患者体内IL-37的表达显著降低,可能导致其无法有效发挥对DC分泌促炎性细胞因子和产生活性氧的抑制作用以及对Th17细胞和Th1细胞分化的调节作用,从而参与到Behcet’s病的发生。第二部分BTLA在Behcet’s病发病机制中的作用研究目的:Behcet’s病是一种严重损害视力的疾病。已有研究报道Th17细胞和Th1细胞的过度激活与此病的发生密切相关。BTLA已被证实在免疫反应中起着重要的调节作用。本研究主要探讨BTLA的活化是否能够调节Behcet’s病中异常的免疫反应,以及它在此病的发病过程中的作用及其可能机制。方法:1.抽取活动期Behcet’s病患者和健康对照的外周血,分离PBMCs。利用磁珠分选CD4+T细胞或CD14+单核细胞,利用流式细胞术将CD4+T细胞分选为BTLAhi和BTLAlo两群细胞。2.利用RT-PCR和流式细胞术检测Behcet’s病患者和健康对照体内PBMCs表面BTLA的表达水平;利用流式细胞术检测Behcet’s病患者和健康对照体内PBMCs不同亚群表面BTLA的表达情况。3.利用流式细胞术检测患者和健康对照体内Th17细胞和Th1细胞比例,以及产生IL-17和IFN-γ的BTLAhiCD4+T细胞和BTLAloCD4+T细胞比例。4.利用BTLA特异性激动剂活化BTLA,并评价其对患者和健康对照体内IL-17、IFN-γ和IL-22分泌的影响和对Th17细胞、Th1细胞比例的影响。5.利用流式细胞术和ELISAs检测BTLA活化对DC诱导的Th17细胞和Th1细胞免疫反应的影响。6.利用流式细胞术和ELISAs评价BTLA活化对DC表面标志物的影响及对DC分泌Th17细胞、Th1细胞活化相关细胞因子的影响。结果:1.活动期Behcet’s病患者体内BTLA m RNA及蛋白水平显著低于健康对照;患者体内BTLA在CD4+T细胞表面的表达也明显降低。2.活动期Behcet’s病患者体内Th17细胞和Th1细胞比例均显著高于健康对照,活动期Behcet’s病患者Th17细胞和Th1细胞免疫反应过度活化。3.经anti-CD3和anti-CD28预处理后,患者体内的BTLAloIL-17+细胞比例显著高于正常人,同时BTLAloCD4+T细胞表达更多IL-17和IFN-γ。4.BTLA特异性激动剂活化BTLA后,患者和健康对照的CD4+T细胞分泌的IL-17、IL-22和IFN-γ均显著降低,同时患者和健康对照体内的Th17细胞和Th1细胞比例也明显下降。5.活化BTLA明显抑制DC细胞诱导的Th17细胞和Th1细胞活化,并抑制CD4+T细胞分泌IL-17和IFN-γ。6.活化BTLA显著抑制DC表面与T细胞活化相关的分子CD40的表达,同时抑制DC分泌Th17细胞和Th1细胞活化相关细胞因子IL-1β、IL-6、IL-23和IL-12p70。结论:我们的研究发现,活动性Behcet’s病患者体内BTLA表达降低与Th17细胞和Th1细胞的过度活化密切相关。BTLA的表达降低可能影响其对Th17细胞和Th1细胞免疫反应以及DC功能的调节作用,从而参与到疾病的发生和复发。利用BTLA特异性激动剂活化BTLA可能成为防治Behcet’s病的一个新策略。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R773.9

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