溃疡性结肠炎患者B细胞亚群的变化及相关机制的研究

发布时间：2018-02-01 08:04

本文关键词： 溃疡性结肠炎 B细胞亚群滤泡辅助性T细胞滤泡调节性T细胞　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:溃疡性结肠炎(ulcerative colitis,UC)是一种炎症性肠病(inflammatory bowel disease,IBD),其确切病因和发病过程尚不清楚。目前仍无法治愈UC,疾病多呈慢性病程,且有发展为结肠炎相关癌症(colitis-associated cancer,CAC)的风险。研究UC的发病机制对于寻找新的治疗靶点有重要意义。UC的危险因素包括环境因素,遗传易感性,特别是过度激活的免疫反应。然而B细胞免疫在UC发病机制中的作用知之甚少,活动性UC中B细胞各亚群的频数和作用均未阐明。滤泡调节性T细胞(follicular regulatory T cells,TFR)和滤泡辅助性T细胞(follicular helper T cells,TFH)之间的免疫平衡在调控B细胞反应中起到非常重要的作用。然而UC中TFR细胞以及TFR/TFH比例的变化仍不清楚。目的:我们的目的是研究活动性UC患者B细胞各亚群,TFR细胞,TFH细胞的频数,表型特征和功能的变化,以及相关免疫球蛋白(immunoglobulin,Ig)和细胞因子的水平。此外,我们探究以上各细胞亚群与UC患者疾病活动性指标间潜在的相关性。本研究旨在研究B细胞各亚群在UC发病机制中的作用,及B细胞免疫紊乱的相关机制。方法:由于流式细胞术检测外周血单个核细胞(peripheral blood mononuclear cells,PBMC)中各细胞亚群的频数和具体表型时需要外周血的体积较大,若以一例患者的血液样本完成所有实验可能会对患者造成伤害。因此,我们三部分实验中采用不同的患者及对照组。1.收集23例活动性UC患者和14名健康体检者的肝素钠抗凝外周血,分离血浆及PBMC。流式细胞术检测PBMC中记忆B细胞和浆母细胞各亚群频数的改变。微量样本多指标流式蛋白定量技术(cytometric bead array,CBA)检测血浆中IgG,IgM,IgA的水平。2.收集25例活动性UC患者和15名健康体检者的肝素钠抗凝外周血,分离血浆及PBMC。收集5例活动性UC患者和5名正常对照者结肠组织,分离其黏膜固有层单个核细胞(lamina propria mononuclear cells,LPMC)。流式细胞术检测PBMC和LPMC中Breg细胞各亚群频数改变。酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血浆中IL-10的水平。CBA检测血浆中IgG,IgM,IgA的浓度。流式分选UC患者PBMC中B细胞并进行体外刺激培养,检测其分泌IL-10的水平。3.收集25例活动性UC患者和15名健康体检者的肝素钠抗凝外周血,分离血浆及PBMC。流式细胞仪检测PBMC中B细胞,TFR细胞,TFH细胞和Treg细胞各亚群频数改变。CBA检测血浆中相关细胞因子和Ig的浓度。对于以上所有纳入实验的患者,Mayo临床评分评价疾病活动性,同时检测C反应蛋白(CRP)的浓度以及红细胞沉降率(ESR)。用Spearman相关性检验分析各检测量间潜在的相关性。结果:1.与健康体检者相比,活动期UC患者IgG~+IgD~-CD27~+CD19~+记忆B细胞频数显著减少,CD20-CD19~+浆母细胞亚群显著增加,并且血浆中IgG水平显著增高。Mayo临床指数,CRP或ESR与IgG~+IgD~-CD27~+CD19~+记忆B细胞的频数成负相关,而与各浆母细胞亚群的频数和血浆中IgG浓度成正相关。此外,血浆IgG浓度,各浆母细胞亚群的频数,与IgG~+IgD~-CD27~+CD19~+记忆B细胞的频数成负相关。血浆IgG浓度与CD138+CD38+CD20-CD19~+和IgG~+CD38+CD20-CD19~+浆母细胞的频数成正相关。2.与健康体检者相比,活动期UC患者CD24high CD38high和CD5~+Breg细胞频数都显著减少,同时血浆中IL-10水平显著降低。体外刺激UC患者外周血中分选出的B细胞,其分泌IL-10水平显著下降,并且IL-10+B细胞基本为CD24highCD38high和CD5~+B细胞。然而,Breg细胞中CD95~+的衰竭Breg细胞所占比例显著升高。Mayo临床活动性指数,CRP或ESR与Breg细胞的频数和血浆中IL-10水平成负相关,而与CD95~+的衰竭细胞在Breg细胞中占的比例成正相关。此外,血浆IL-10浓度与CD24high CD38high Breg细胞的频数成正相关。血浆IgG浓度与CD95~+的衰竭细胞在Breg细胞中占的比例成正相关。3.与健康体检者相比,活动期UC患者外周血Foxp3+CXCR5~+TFR细胞,IL-10+Foxp3+CXCR5~+TFR细胞亚群和Treg细胞的频数显著减少,而Foxp3-CXCR5~+TFH细胞和IL-21+Foxp3-CXCR5~+TFH细胞亚群频数显著增多,同时UC患者血浆中IL-10水平下降,IL-21和IgG水平上升。Mayo临床指数,CRP或ESR与TFR细胞的频数,尤其是TFR/TFH比值成负相关;而与TFH细胞和血浆中IL-21水平成正相关。此外,IgG~+浆母细胞的频数与TFR细胞的频数成负相关。血浆IgG浓度与TFR/TFH比值成负相关。结论:1.活动性UC患者B细胞反应过度活跃,Breg细胞的免疫调节功能衰竭。以上B细胞亚群的紊乱参与了UC的发病过程。2.活动性UC患者TFR和TFH细胞的变化导致了免疫激活和免疫耐受之间的平衡失调,促使B细胞免疫过度激活,参与了UC的发病机制。
[Abstract]:Background: ulcerative colitis (ulcerative colitis UC) is a kind of inflammatory bowel disease (inflammatory bowel, disease, IBD), its etiology and pathogenesis is unclear. At present there is no way to cure UC, disease is a chronic disease, and for the development of colitis associated cancer (colitis-associated, cancer, CAC) the incidence of risk. Study of the mechanism of UC is important risk factors of.UC include environmental factors, genetic susceptibility to search for new treatment targets, especially the immune response. However, excessive activation of B cell immunity in the pathogenesis of UC are poorly understood, and the frequency of B cell activity in UC subsets were not clarify. Follicular regulatory T cells (follicular regulatory T cells, TFR) and T follicular helper cells (follicular helper T cells, TFH) immune balance plays a very important role in the regulation of B cell responses. However, UC Changes in TFR cells and the ratio of TFR/TFH is still unclear. Objective: our aim was to study the activity of UC in patients with B cell subsets, TFR cells, the frequency of TFH cells, the phenotype and function, and the associated immunoglobulin (immunoglobulin, Ig) and cytokine levels. In addition, we explore the potential relevance of these cell subsets and UC disease activity index. The purpose of this study is to study the B cell subsets in UC pathogenesis, and related mechanism in B cell immune disorders. Methods: the flow cytometry of peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC the frequency in each cell subsets) and specific phenotype to peripheral blood volume is larger, if a patient completed all experimental blood samples may cause harm to patients. Therefore, the three part of our experiment with different Heparin sodium anticoagulation of patients and the control group.1. was collected from 23 patients with active UC patients and 14 healthy persons in peripheral blood plasma separation and PBMC. flow cytometry PBMC memory B cells and plasma cell subsets frequency change. Trace sample multivariate flow cytometry protein quantitative technique (cytometric bead array, CBA) detection of plasma IgG, IgM,.2., IgA level of heparin sodium anticoagulation collected from 25 patients with active UC patients and 15 healthy persons in peripheral blood plasma separation and PBMC. collected 5 cases of active UC patients and 5 normal controls colon tissue of the mucosal layer with solid separation of mononuclear cells (lamina propria mononuclear cells, LPMC). The change of Breg cell cytometry was used to detect PBMC and LPMC subsets in frequency flow. Enzyme linked immunosorbent assay (enzyme-linked immunosorbent, assay, ELISA) detection of plasma.CBA level of IL-10 in the detection of plasma IgG, IgM, IgA. The degree of B type UC cells. Flow separation in PBMC patients and cultured in vitro, heparin to detect the secretion level of IL-10.3. was collected from 25 patients with active UC patients and 15 healthy persons in peripheral blood plasma separation and PBMC. flow cytometry to detect PBMC in B cells, TFR cells, TFH cells and Treg cell subsets of frequency change related cytokines Ig and.CBA concentrations in plasma were detected for all the patients included in the experiment, Mayo clinical evaluation of disease activity, simultaneous detection of C reactive protein (CRP) concentration and erythrocyte sedimentation rate (ESR). The correlation analysis between the amount of testing for potential Spearman correlation test. Results: 1. compared with healthy subjects, patients with active IgG~+IgD~-CD27~+CD19~+ UC memory B cell frequency was significantly reduced, CD20-CD19~+ plasmablast subsets increased significantly, and the level of plasma IgG was significantly higher in clinical.Mayo The number of CRP or ESR and IgG~+IgD~-CD27~+CD19~+, the frequency of memory B cells is negatively correlated, but positively correlated with the plasma frequency and plasmablast subsets in IgG concentration. In addition, the plasma concentration of IgG, the frequency of plasmablast subsets, negative correlation with the frequency of IgG~+IgD~-CD27~+CD19~+ memory B cells into plasma frequency compared. The concentration of IgG and CD138+CD38+CD20-CD19~+ and IgG~+CD38+CD20-CD19~+ plasmablast are positively correlated with.2. in healthy subjects, patients with active UC CD24high CD38high and the frequency of CD5~+Breg cells decreased significantly, while the plasma IL-10 level was significantly decreased. UC were separated from in vitro stimulation of B cells in peripheral blood, the secretion of IL-10 and IL-10+B were significantly decreased. The basic cell for CD24highCD38high and CD5~+B cells. However, for CD95~+ Breg cells in Breg cells was significantly increased the proportion of failure index of clinical activity of.Mayo, CRP or ESR and B The level of IL-10 into reg cells and plasma frequency in negative correlation with the CD95~+ failure in Breg cells proportion was positively correlated. In addition, the concentration of plasma IL-10 and CD24high frequency CD38high Breg cells are positively correlated. Compared the concentration of plasma IgG and CD95~+ failure in Breg cells the percentage of positive correlation between.3. and healthy subjects, Foxp3+CXCR5~+TFR cells in the peripheral blood of patients with active UC, frequency and Treg cell subsets of IL-10+Foxp3+CXCR5~+TFR cells decreased significantly, while Foxp3-CXCR5~+TFH cells and IL-21+Foxp3-CXCR5~+TFH cell subsets in frequency increased significantly, while the plasma IL-10 level in patients with UC decreased, IL-21 and IgG increased the level of.Mayo clinical index, the frequency of CRP or ESR and TFR cells, especially TFR/TFH ratio was negatively correlated; and TFH cells and plasma IL-21 levels were positively correlated. In addition, the frequency of TFR cells and IgG~+ of plasmablast The frequency of negative correlation. The concentration of plasma IgG and TFR/TFH ratio are negatively correlated. Conclusion: 1. B cells in patients with UC over active immune Breg cell regulation failure. These disorders of B cell subsets in the pathogenesis of.2. UC changes of patients with UC TFR and TFH cells led to between immune activation and immune tolerance imbalance, promote B cell immune activation involved in the pathogenesis of UC.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R574.62

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