MPO-ANCA相關(guān)性血管炎患者外周血濾泡輔助性T細胞及白介素-21水平的變化及意義
發(fā)布時間：2018-08-08 14:20
【摘要】：背景ANCA相關(guān)性血管炎(Antineutrophil cytoplasmic autoantibody associated vasculitis,AAV)是一類累及全身小血管的自身免疫性血管炎,主要包括肉芽腫性多血管炎(Granulomatosis with polyangiitis,GPA)、顯微鏡下多血管炎(Microscopic polyangiitis,MPA)及嗜酸性肉芽腫性多血管炎(Eosinophilic granulomatosis with polyangiitis,EGPA)。ANCA是該類血管炎的主要血清學標志物,其特異性抗原主要有髓過氧化物酶(Myeloperoxidase,MPO)和蛋白酶-3(Proteinase-3,PR-3),與歐美等國家不同,我國MPO-AAV比PR3-AAV更為常見。此外,MPO-AAV發(fā)病過程隱匿不易發(fā)現(xiàn),而且病情進展迅速,受累臟器多,死亡率高,因此加強對MPO-AAV發(fā)病機制的研究十分必要。MPO-AAV的確切發(fā)病機制尚不明確,研究發(fā)現(xiàn)MPO-ANCA可能參與了MPO-AAV發(fā)病。MPO-ANCA做為一種自身抗體,由漿細胞產(chǎn)生,B細胞分化為漿細胞及漿細胞分泌抗體的過程受到自身免疫系統(tǒng)的調(diào)控。近年來一種定位于淋巴濾泡的新型T細胞亞群,濾泡輔助性T細胞(Follicular helper T cells,Tfh)逐漸被認識。該細胞特異性表達CXC趨化因子受體5(CXC-chemokine receptor 5,CXCR5)、可誘導的共刺激分子(Inducible co-stimulator,ICOS)、程序性死亡因子-1(Programmed cell death protein 1,PD-1)等,并分泌白介素-21(Interleukin,IL-21),主要作用是輔助B細胞分化為成熟的漿細胞及產(chǎn)生抗體。研究發(fā)現(xiàn),Tfh細胞與多種自身免疫病有關(guān),而Tfh在MPO-AAV中的作用尚未見研究報道。目的探討MPO-AAV患者的外周血濾泡輔助性T細胞及白介素-21水平的變化及其與疾病活動度、臨床損害間的關(guān)系。方法選擇40例初發(fā)的、未接受治療的MPO-AAV患者及30例健康人的新鮮抗凝血,先制備單個核細胞懸液(Peripheral blood mononuclear cells,PBMCs),運用流式細胞術(shù)(flow cytometry,FCM)檢測血管炎患者和健康人外周血Tfh的表達,將CXCR5+CD4+T細胞定義為循環(huán)血Tfh細胞(c Tfh細胞),同時檢測c Tfh細胞表面分子ICOS及PD-1表達的細胞百分率及平均熒光強度(Mean fluorescence intensity,MFI)。運用酶聯(lián)免疫吸附測定(enzyme-linked immunosorbent assay,ELISA)方法檢測患者和健康人血清IL-21水平及患者MPO-ANCA水平。詳細記錄患者的臨床指標、臟器損害情況及伯明翰血管炎活動度積分(Birmingham Vasculitis Activity Score-version 3,BVAS-V3)。并比較各指標在血管炎組及健康對照組兩組之間的表達差異,分析患者組各指標之間的相關(guān)性,并探討其與血管炎疾病活動度及臟器損害間的關(guān)系。結(jié)果1.一般臨床資料:MPO-AAV組共40例,其中男17例,女23例,年齡23~85(64.43±13.36)歲,病程0.5~240(3[1,48])月,血沉7~140(69.31±36.33)mm/h,C反應蛋白(CRP)0.50~176.92(59.33±49.32)mg/L,BVAS值5~37(18.93±5.89)分;健康對照組共30例,其中男13例,女17例,年齡38~79(62.70±11.30)歲。兩組之間性別及年齡分布均無明顯差異(p0.05),兩組數(shù)據(jù)具有可比性。2.FCM結(jié)果:MPO-AAV組c Tfh、ICOS+c Tfh及PD-1+c Tfh細胞的百分率顯著高于健康對照組(25.79%±3.72%vs 19.98%±4.72%,p0.001;1.83%±1.06%vs 0.82%±0.60%,p0.001;10.10%±2.85%vs 8.17%±2.66%,p=0.018)。與健康對照組相比,MPO-AAV組c Tfh細胞表面ICOS與PD-1的表達強度MFI均顯著升高(59.16±10.12 vs 48.88±7.36,p0.001;532.71±81.14 vs 449.15±65.99,p0.001);3.ELISA結(jié)果:MPO-AAV組外周血血清IL-21水平顯著高于健康對照組(Z=-4.12,p=0.005);MPO-AAV組外周血血清MPO-ANCA水平(OD值)為0.352[0.248,0.433];4.MPO-AAV組各檢測指標之間的相關(guān)性分析:MPO-AAV患者c Tfh細胞百分率分別與血清IL-21及血清MPO-ANCA水平均呈正相關(guān)(r=0.497,p0.01;r=0.736,p0.01)。c Tfh細胞ICOS的MFI分別與血清IL-21、MPO-ANCA水平呈顯著正相關(guān)(r=0.586,p0.01;r=0.607,p0.01)。血清IL-21水平與血清MPO-ANCA水平呈顯著正相關(guān)(r=0.668,p0.01)。而ICOS+c Tfh、PD-1+c Tfh細胞百分率及c Tfh細胞PD-1的MFI與血清IL-21、MPO-ANCA水平均無相關(guān)性。5.MPO-AAV組各檢測指標與臨床指標之間的相關(guān)性分析:MPO-AAV患者BVAS評分別與c Tfh細胞百分率、c Tfh細胞ICOS的MFI及血清MPO-ANCA水平呈正相關(guān)(r=0.564,p0.01;r=0.513,p0.01;r=0.358,p0.05)。6.MPO-AAV患者中,有腎臟損害組c Tfh細胞百分率及c Tfh細胞ICOS的MFI均高于無腎臟損害組,差異有統(tǒng)計學意義(p0.05)。有肺損害患者與無肺損害患者相比,各主要實驗室檢測指標間無明顯差異。7.MPO-AAV患者中腎BVAS及肺BVAS分別與各指標之間的相關(guān)性分析,結(jié)果顯示,患者腎臟BVAS與c Tfh細胞及其表面分子ICOS表達強度(MFI)呈正相關(guān)(r=0.393,p0.05;r=0.389,p0.05),與其他各指標之間均未見相關(guān)性�；颊叻尾緽VAS與所有指標之間均未見相關(guān)性。結(jié)論1.MPO-AAV患者c Tfh及其表面分子ICOS和PD-1的表達水平、IL-21水平均高于對照組,提示MPO-AAV患者中存在c Tfh細胞數(shù)量及功能異常;2.MPO-AAV患者c Tfh、血清IL-21及血清MPO-ANCA水平兩兩之間均呈正相關(guān),提示c Tfh可能通過輔助B細胞產(chǎn)生致病性抗體MPO-ANCA,參與MPO-AAV的發(fā)病,這一過程可能是通過IL-21實現(xiàn)的;3.MPO-AAV患者c Tfh數(shù)目升高,而且c Tfh細胞百分率及ICOS的MFI均與BVAS評分呈正相關(guān),提示c Tfh細胞數(shù)量及功能可能參與MPO-AAV的發(fā)病和病情進展。4.MPO-AAV患者中有腎臟損害與c Tfh細胞之間的研究結(jié)果,提示MPO-AAV中c Tfh細胞可能與腎臟損害的發(fā)生和發(fā)展有關(guān)。
[Abstract]:Background ANCA related vasculitis (Antineutrophil cytoplasmic autoantibody associated vasculitis, AAV) is a kind of autoimmune vasculitis involving the small vessels of the whole body, including granulomatous vasculitis (Granulomatosis with polyangiitis, GPA), microvasculitis (Microscopic), and eosinophilic meat under microscope. Eosinophilic granulomatosis with polyangiitis (EGPA).ANCA is the main serological marker of this kind of vasculitis. Its specific antigens mainly include myeloperoxidase (Myeloperoxidase, MPO) and protease -3 (Proteinase-3, PR-3). Unlike countries like Europe and America, our MPO-AAV is more common than those in our country. The pathogenesis is not easy to be found, and the disease is progressing rapidly, many organs are involved and the death rate is high. Therefore, it is necessary to strengthen the study of the pathogenesis of MPO-AAV. It is not clear that the exact pathogenesis of.MPO-AAV is not clear. The study found that MPO-ANCA may be involved in the pathogenesis of MPO-AAV as a kind of autoantibody, produced by plasma cells and B cells differentiated into The process of secreting antibodies in plasma cells and plasma cells is regulated by the autoimmune system. In recent years a new type of T cell subgroup located in lymphoid follicles and follicular auxiliary T cells (Follicular helper T cells, Tfh) are gradually recognized. The cells specifically express CXC chemokine receptor 5 (CXC-chemokine receptor 5, CXCR5), and can induce co - induction. The stimulator (Inducible co-stimulator, ICOS), the programmed death factor -1 (Programmed cell death protein 1, PD-1), and the secretion of interleukin -21 (Interleukin, IL-21). The main function is to assist the differentiated cells to differentiate into mature plasma cells and produce antibodies. Objective to investigate the changes in peripheral blood follicle assisted T cells and interleukin -21 levels in the peripheral blood of MPO-AAV patients and their relationship with the degree of disease activity and clinical damage. Methods 40 cases of primary, untreated MPO-AAV and 30 healthy individuals were selected to prepare mononuclear cell suspension first (Peripheral Blood mononuclear cells, PBMCs), using flow cytometry (flow cytometry, FCM) to detect the expression of Tfh in peripheral blood of patients with vasculitis and healthy people. CXCR5+CD4+T cells are defined as circulating blood Tfh cells (C Tfh cells), and the percentage of cell surface molecules and the average fluorescence intensity of the cells are detected. Intensity, MFI). Using enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) to detect serum IL-21 level and patient MPO-ANCA level in patients and healthy people. The clinical indicators, organ damage and Bermingham vasculitis activity (Birmingham Vasculitis Activity Score-version 3) were recorded in detail. 3) and compared the expression difference between the two groups in the vasculitis group and the healthy control group, analyzed the correlation between the indexes of the patients, and discussed the relationship with the activity of vasculitis and the organ damage. Results 1. general clinical data: 40 cases in group MPO-AAV, including 17 male, 23 female, age 23~85 (64.43 + 13.36), and 0. course of disease 0.. 5~240 (3[1,48]) month, erythrocyte sedimentation rate 7~140 (69.31 + 36.33) mm/h, C reactive protein (CRP) 0.50~176.92 (59.33 + 49.32) mg/L, BVAS 5~37 (18.93 + 5.89), 30 cases in healthy control group, including 13 men, 17 women, age 38~79 (62.70 + 11.30) years. There was no significant difference between sex and age distribution between two groups (P0.05), two group data have comparability results The percentage of C Tfh, ICOS+c Tfh and PD-1+c Tfh cells in the MPO-AAV group was significantly higher than that in the healthy control group (25.79% + 3.72%vs 19.98% + 4.72%, p0.001; 1.83% + 1.06%vs 0.82% + 0.60%, p0.001; 10.10% + 8.17% + 2.66%, respectively). 10.12 vs 48.88 + 7.36, p0.001, 532.71 + 81.14 vs 449.15 + 65.99, p0.001); 3.ELISA results: the serum serum IL-21 level of MPO-AAV group was significantly higher than that of the healthy control group (Z=-4.12, p=0.005); the MPO-ANCA level of the peripheral blood serum in the MPO-AAV group was 0.352[0.248,0.433]. The percentage of Tfh cells was positively correlated with the level of serum IL-21 and serum MPO-ANCA (r=0.497, P0.01; r=0.736, P0.01).C Tfh cell ICOS MFI respectively. The percentage of D-1+c Tfh cells and the MFI of PD-1 in C Tfh cells and serum IL-21, MPO-ANCA level were not related to the correlation analysis between the indexes of.5.MPO-AAV group and the clinical indexes. In 358, P0.05).6.MPO-AAV patients, the percentage of C Tfh cells in the kidney damage group and the MFI of ICOS in C Tfh cells were higher than those in the non renal damage group, the difference was statistically significant (P0.05). There was no significant difference between the patients with lung damage and the non lung injury patients, and there was no significant difference between the main laboratory test indexes of the major laboratory tests, and the renal BVAS and lung BVAS were respectively and each finger in the.7.MPO-AAV patients. The correlation analysis between the markers showed that the renal BVAS of the patient was positively correlated with the ICOS expression intensity (MFI) of the C Tfh cells and their surface molecules (r=0.393, P0.05; r=0.389, P0.05), and had no correlation with the other indexes. There was no correlation between the lung BVAS and all the indexes. The level of expression of PD-1 and the level of IL-21 were higher than that of the control group, suggesting that there was a number of C Tfh cells and abnormal function in MPO-AAV patients, and there was a positive correlation between C Tfh, serum IL-21 and serum MPO-ANCA level in 2.MPO-AAV patients, suggesting that C Tfh may be involved in the pathogenesis of pathogenic antibodies by assisting the cells. The number of C Tfh in patients with 3.MPO-AAV increased, and the percentage of C Tfh cells and the MFI of ICOS were positively correlated with the BVAS score, suggesting that the number and function of C Tfh cells may be involved in the pathogenesis and progression of MPO-AAV. The cells may be related to the occurrence and development of renal damage.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R593.2
本文編號：2172061
[Abstract]:Background ANCA related vasculitis (Antineutrophil cytoplasmic autoantibody associated vasculitis, AAV) is a kind of autoimmune vasculitis involving the small vessels of the whole body, including granulomatous vasculitis (Granulomatosis with polyangiitis, GPA), microvasculitis (Microscopic), and eosinophilic meat under microscope. Eosinophilic granulomatosis with polyangiitis (EGPA).ANCA is the main serological marker of this kind of vasculitis. Its specific antigens mainly include myeloperoxidase (Myeloperoxidase, MPO) and protease -3 (Proteinase-3, PR-3). Unlike countries like Europe and America, our MPO-AAV is more common than those in our country. The pathogenesis is not easy to be found, and the disease is progressing rapidly, many organs are involved and the death rate is high. Therefore, it is necessary to strengthen the study of the pathogenesis of MPO-AAV. It is not clear that the exact pathogenesis of.MPO-AAV is not clear. The study found that MPO-ANCA may be involved in the pathogenesis of MPO-AAV as a kind of autoantibody, produced by plasma cells and B cells differentiated into The process of secreting antibodies in plasma cells and plasma cells is regulated by the autoimmune system. In recent years a new type of T cell subgroup located in lymphoid follicles and follicular auxiliary T cells (Follicular helper T cells, Tfh) are gradually recognized. The cells specifically express CXC chemokine receptor 5 (CXC-chemokine receptor 5, CXCR5), and can induce co - induction. The stimulator (Inducible co-stimulator, ICOS), the programmed death factor -1 (Programmed cell death protein 1, PD-1), and the secretion of interleukin -21 (Interleukin, IL-21). The main function is to assist the differentiated cells to differentiate into mature plasma cells and produce antibodies. Objective to investigate the changes in peripheral blood follicle assisted T cells and interleukin -21 levels in the peripheral blood of MPO-AAV patients and their relationship with the degree of disease activity and clinical damage. Methods 40 cases of primary, untreated MPO-AAV and 30 healthy individuals were selected to prepare mononuclear cell suspension first (Peripheral Blood mononuclear cells, PBMCs), using flow cytometry (flow cytometry, FCM) to detect the expression of Tfh in peripheral blood of patients with vasculitis and healthy people. CXCR5+CD4+T cells are defined as circulating blood Tfh cells (C Tfh cells), and the percentage of cell surface molecules and the average fluorescence intensity of the cells are detected. Intensity, MFI). Using enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) to detect serum IL-21 level and patient MPO-ANCA level in patients and healthy people. The clinical indicators, organ damage and Bermingham vasculitis activity (Birmingham Vasculitis Activity Score-version 3) were recorded in detail. 3) and compared the expression difference between the two groups in the vasculitis group and the healthy control group, analyzed the correlation between the indexes of the patients, and discussed the relationship with the activity of vasculitis and the organ damage. Results 1. general clinical data: 40 cases in group MPO-AAV, including 17 male, 23 female, age 23~85 (64.43 + 13.36), and 0. course of disease 0.. 5~240 (3[1,48]) month, erythrocyte sedimentation rate 7~140 (69.31 + 36.33) mm/h, C reactive protein (CRP) 0.50~176.92 (59.33 + 49.32) mg/L, BVAS 5~37 (18.93 + 5.89), 30 cases in healthy control group, including 13 men, 17 women, age 38~79 (62.70 + 11.30) years. There was no significant difference between sex and age distribution between two groups (P0.05), two group data have comparability results The percentage of C Tfh, ICOS+c Tfh and PD-1+c Tfh cells in the MPO-AAV group was significantly higher than that in the healthy control group (25.79% + 3.72%vs 19.98% + 4.72%, p0.001; 1.83% + 1.06%vs 0.82% + 0.60%, p0.001; 10.10% + 8.17% + 2.66%, respectively). 10.12 vs 48.88 + 7.36, p0.001, 532.71 + 81.14 vs 449.15 + 65.99, p0.001); 3.ELISA results: the serum serum IL-21 level of MPO-AAV group was significantly higher than that of the healthy control group (Z=-4.12, p=0.005); the MPO-ANCA level of the peripheral blood serum in the MPO-AAV group was 0.352[0.248,0.433]. The percentage of Tfh cells was positively correlated with the level of serum IL-21 and serum MPO-ANCA (r=0.497, P0.01; r=0.736, P0.01).C Tfh cell ICOS MFI respectively. The percentage of D-1+c Tfh cells and the MFI of PD-1 in C Tfh cells and serum IL-21, MPO-ANCA level were not related to the correlation analysis between the indexes of.5.MPO-AAV group and the clinical indexes. In 358, P0.05).6.MPO-AAV patients, the percentage of C Tfh cells in the kidney damage group and the MFI of ICOS in C Tfh cells were higher than those in the non renal damage group, the difference was statistically significant (P0.05). There was no significant difference between the patients with lung damage and the non lung injury patients, and there was no significant difference between the main laboratory test indexes of the major laboratory tests, and the renal BVAS and lung BVAS were respectively and each finger in the.7.MPO-AAV patients. The correlation analysis between the markers showed that the renal BVAS of the patient was positively correlated with the ICOS expression intensity (MFI) of the C Tfh cells and their surface molecules (r=0.393, P0.05; r=0.389, P0.05), and had no correlation with the other indexes. There was no correlation between the lung BVAS and all the indexes. The level of expression of PD-1 and the level of IL-21 were higher than that of the control group, suggesting that there was a number of C Tfh cells and abnormal function in MPO-AAV patients, and there was a positive correlation between C Tfh, serum IL-21 and serum MPO-ANCA level in 2.MPO-AAV patients, suggesting that C Tfh may be involved in the pathogenesis of pathogenic antibodies by assisting the cells. The number of C Tfh in patients with 3.MPO-AAV increased, and the percentage of C Tfh cells and the MFI of ICOS were positively correlated with the BVAS score, suggesting that the number and function of C Tfh cells may be involved in the pathogenesis and progression of MPO-AAV. The cells may be related to the occurrence and development of renal damage.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R593.2
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