本文關(guān)鍵詞： 潰瘍性結(jié)腸炎 B細(xì)胞亞群 濾泡輔助性T細(xì)胞 濾泡調(diào)節(jié)性T細(xì)胞　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：背景:潰瘍性結(jié)腸炎(ulcerative colitis,UC)是一種炎癥性腸病(inflammatory bowel disease,IBD),其確切病因和發(fā)病過程尚不清楚。目前仍無法治愈UC,疾病多呈慢性病程,且有發(fā)展為結(jié)腸炎相關(guān)癌癥(colitis-associated cancer,CAC)的風(fēng)險(xiǎn)。研究UC的發(fā)病機(jī)制對(duì)于尋找新的治療靶點(diǎn)有重要意義。UC的危險(xiǎn)因素包括環(huán)境因素,遺傳易感性,特別是過度激活的免疫反應(yīng)。然而B細(xì)胞免疫在UC發(fā)病機(jī)制中的作用知之甚少,活動(dòng)性UC中B細(xì)胞各亞群的頻數(shù)和作用均未闡明。濾泡調(diào)節(jié)性T細(xì)胞(follicular regulatory T cells,TFR)和濾泡輔助性T細(xì)胞(follicular helper T cells,TFH)之間的免疫平衡在調(diào)控B細(xì)胞反應(yīng)中起到非常重要的作用。然而UC中TFR細(xì)胞以及TFR/TFH比例的變化仍不清楚。目的:我們的目的是研究活動(dòng)性UC患者B細(xì)胞各亞群,TFR細(xì)胞,TFH細(xì)胞的頻數(shù),表型特征和功能的變化,以及相關(guān)免疫球蛋白(immunoglobulin,Ig)和細(xì)胞因子的水平。此外,我們探究以上各細(xì)胞亞群與UC患者疾病活動(dòng)性指標(biāo)間潛在的相關(guān)性。本研究旨在研究B細(xì)胞各亞群在UC發(fā)病機(jī)制中的作用,及B細(xì)胞免疫紊亂的相關(guān)機(jī)制。方法:由于流式細(xì)胞術(shù)檢測(cè)外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells,PBMC)中各細(xì)胞亞群的頻數(shù)和具體表型時(shí)需要外周血的體積較大,若以一例患者的血液樣本完成所有實(shí)驗(yàn)可能會(huì)對(duì)患者造成傷害。因此,我們?nèi)糠謱?shí)驗(yàn)中采用不同的患者及對(duì)照組。1.收集23例活動(dòng)性UC患者和14名健康體檢者的肝素鈉抗凝外周血,分離血漿及PBMC。流式細(xì)胞術(shù)檢測(cè)PBMC中記憶B細(xì)胞和漿母細(xì)胞各亞群頻數(shù)的改變。微量樣本多指標(biāo)流式蛋白定量技術(shù)(cytometric bead array,CBA)檢測(cè)血漿中IgG,IgM,IgA的水平。2.收集25例活動(dòng)性UC患者和15名健康體檢者的肝素鈉抗凝外周血,分離血漿及PBMC。收集5例活動(dòng)性UC患者和5名正常對(duì)照者結(jié)腸組織,分離其黏膜固有層單個(gè)核細(xì)胞(lamina propria mononuclear cells,LPMC)。流式細(xì)胞術(shù)檢測(cè)PBMC和LPMC中Breg細(xì)胞各亞群頻數(shù)改變。酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay,ELISA)檢測(cè)血漿中IL-10的水平。CBA檢測(cè)血漿中IgG,IgM,IgA的濃度。流式分選UC患者PBMC中B細(xì)胞并進(jìn)行體外刺激培養(yǎng),檢測(cè)其分泌IL-10的水平。3.收集25例活動(dòng)性UC患者和15名健康體檢者的肝素鈉抗凝外周血,分離血漿及PBMC。流式細(xì)胞儀檢測(cè)PBMC中B細(xì)胞,TFR細(xì)胞,TFH細(xì)胞和Treg細(xì)胞各亞群頻數(shù)改變。CBA檢測(cè)血漿中相關(guān)細(xì)胞因子和Ig的濃度。對(duì)于以上所有納入實(shí)驗(yàn)的患者,Mayo臨床評(píng)分評(píng)價(jià)疾病活動(dòng)性,同時(shí)檢測(cè)C反應(yīng)蛋白(CRP)的濃度以及紅細(xì)胞沉降率(ESR)。用Spearman相關(guān)性檢驗(yàn)分析各檢測(cè)量間潛在的相關(guān)性。結(jié)果:1.與健康體檢者相比,活動(dòng)期UC患者IgG~+IgD~-CD27~+CD19~+記憶B細(xì)胞頻數(shù)顯著減少,CD20-CD19~+漿母細(xì)胞亞群顯著增加,并且血漿中IgG水平顯著增高。Mayo臨床指數(shù),CRP或ESR與IgG~+IgD~-CD27~+CD19~+記憶B細(xì)胞的頻數(shù)成負(fù)相關(guān),而與各漿母細(xì)胞亞群的頻數(shù)和血漿中IgG濃度成正相關(guān)。此外,血漿IgG濃度,各漿母細(xì)胞亞群的頻數(shù),與IgG~+IgD~-CD27~+CD19~+記憶B細(xì)胞的頻數(shù)成負(fù)相關(guān)。血漿IgG濃度與CD138+CD38+CD20-CD19~+和IgG~+CD38+CD20-CD19~+漿母細(xì)胞的頻數(shù)成正相關(guān)。2.與健康體檢者相比,活動(dòng)期UC患者CD24high CD38high和CD5~+Breg細(xì)胞頻數(shù)都顯著減少,同時(shí)血漿中IL-10水平顯著降低。體外刺激UC患者外周血中分選出的B細(xì)胞,其分泌IL-10水平顯著下降,并且IL-10+B細(xì)胞基本為CD24highCD38high和CD5~+B細(xì)胞。然而,Breg細(xì)胞中CD95~+的衰竭Breg細(xì)胞所占比例顯著升高。Mayo臨床活動(dòng)性指數(shù),CRP或ESR與Breg細(xì)胞的頻數(shù)和血漿中IL-10水平成負(fù)相關(guān),而與CD95~+的衰竭細(xì)胞在Breg細(xì)胞中占的比例成正相關(guān)。此外,血漿IL-10濃度與CD24high CD38high Breg細(xì)胞的頻數(shù)成正相關(guān)。血漿IgG濃度與CD95~+的衰竭細(xì)胞在Breg細(xì)胞中占的比例成正相關(guān)。3.與健康體檢者相比,活動(dòng)期UC患者外周血Foxp3+CXCR5~+TFR細(xì)胞,IL-10+Foxp3+CXCR5~+TFR細(xì)胞亞群和Treg細(xì)胞的頻數(shù)顯著減少,而Foxp3-CXCR5~+TFH細(xì)胞和IL-21+Foxp3-CXCR5~+TFH細(xì)胞亞群頻數(shù)顯著增多,同時(shí)UC患者血漿中IL-10水平下降,IL-21和IgG水平上升。Mayo臨床指數(shù),CRP或ESR與TFR細(xì)胞的頻數(shù),尤其是TFR/TFH比值成負(fù)相關(guān);而與TFH細(xì)胞和血漿中IL-21水平成正相關(guān)。此外,IgG~+漿母細(xì)胞的頻數(shù)與TFR細(xì)胞的頻數(shù)成負(fù)相關(guān)。血漿IgG濃度與TFR/TFH比值成負(fù)相關(guān)。結(jié)論:1.活動(dòng)性UC患者B細(xì)胞反應(yīng)過度活躍,Breg細(xì)胞的免疫調(diào)節(jié)功能衰竭。以上B細(xì)胞亞群的紊亂參與了UC的發(fā)病過程。2.活動(dòng)性UC患者TFR和TFH細(xì)胞的變化導(dǎo)致了免疫激活和免疫耐受之間的平衡失調(diào),促使B細(xì)胞免疫過度激活,參與了UC的發(fā)病機(jī)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R574.62

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