本文關(guān)鍵詞：濾泡輔助性T細(xì)胞（Tfh）與自身免疫性皰病的相關(guān)性研究，由筆耕文化傳播整理發(fā)布。

        自身免疫性皰�。╝utoimmune bullous diseases, ABD）是一類臨床常見的自身免疫性疾病，臨床特征為皮膚紅斑、水皰、大皰、糜爛，多發(fā)于中老年人，病情頑固且易反復(fù)發(fā)作。ABD包括兩大類：表皮內(nèi)皰病和表皮下皰病，前者以天皰瘡（pemphigus）最為多見，后者則以大皰性類天皰瘡（bullouspemphigoid，BP）為典型代表[1]。目前由致病性自身抗體介導(dǎo)的皰病免疫學(xué)發(fā)病機(jī)制已得到公認(rèn)，致病性自身抗體與靶抗原結(jié)合，引起一系列免疫炎癥反應(yīng)，如激活補(bǔ)體、炎細(xì)胞浸潤(rùn)、釋放蛋白酶或溶酶體酶等，破壞在表皮細(xì)胞間或真表皮間黏附中起重要作用的結(jié)構(gòu)分子，或直接影響結(jié)構(gòu)分子的功能，導(dǎo)致表皮內(nèi)或表皮下水皰、大皰的發(fā)生[2]。近年來該類疾病免疫學(xué)發(fā)病機(jī)理的研究取得了很大進(jìn)展，明確尋常型天皰瘡（PV）和落葉型天皰瘡（PF）的主要靶抗原分別為橋粒芯糖蛋白3（Dsg3）和橋粒芯糖蛋白1（Dsg1），BP的主要靶抗原為大皰性類天皰瘡抗原1（BPAG1，BP230）和大皰性類天皰瘡抗原2（BPAG2，BP180）[3]，但是致病性自身抗體的來源及其產(chǎn)生機(jī)理尚不清楚。以往研究表明，T細(xì)胞依賴抗原（TD抗原）刺激B細(xì)胞產(chǎn)生抗體必需CD4+輔助性T細(xì)胞（Th）的輔助�？贵w分泌細(xì)胞產(chǎn)生于外周淋巴器官（淋巴結(jié)、脾臟、扁桃體和派氏集合淋巴結(jié)等）的生發(fā)中心（Germinal Centers, GC），并在那里發(fā)生體細(xì)胞高頻突變、抗體類別轉(zhuǎn)換和高親和力的選擇。Th在GC的形成，選擇高親和力B細(xì)胞分化為記憶細(xì)胞或漿細(xì)胞，以及維持長(zhǎng)時(shí)間的體液免疫應(yīng)答等過程中都發(fā)揮著重要作用[4]。近期研究證實(shí)，體液免疫中輔助B細(xì)胞產(chǎn)生抗體的T細(xì)胞亞群為濾泡輔助性T細(xì)胞（T follicular helper cells,Tfh）。Tfh定位于淋巴濾泡，轉(zhuǎn)錄因子為Bcl-6，主要通過分泌白細(xì)胞介素21（IL-21）發(fā)揮作用，其膜表面高表達(dá)CXCR5、ICOS、PD-1、CD40L等，通過與B細(xì)胞表面相應(yīng)的配體或受體相互結(jié)合輔助B細(xì)胞產(chǎn)生高親和力抗體[5]。Tfh水平的異常與自身免疫性疾病的發(fā)病密切相關(guān)。在多種自身免疫性疾病如系統(tǒng)性紅斑狼瘡（SLE）、類風(fēng)濕性關(guān)節(jié)炎（RA）、重癥肌無力（MG）等患者或動(dòng)物模型中均發(fā)現(xiàn)GC的形成和自身抗體的產(chǎn)生涉及Tfh細(xì)胞的異常。在小鼠模型中，阻斷Tfh重要相關(guān)分子的功能，如ICOS、CD40L、IL-21等，則導(dǎo)致自身抗體產(chǎn)生下降[6-10]。鑒于目前自身免疫性皰病的自身抗體來源及產(chǎn)生機(jī)制尚不明確，而Tfh的主要功能是輔助B細(xì)胞產(chǎn)生高親和力抗體，我們推測(cè)Tfh參與介導(dǎo)了自身免疫性皰病自身抗體的產(chǎn)生。本實(shí)驗(yàn)通過檢測(cè)天皰瘡和大皰性類天皰瘡患者外周血中Tfh細(xì)胞水平，初步探索Tfh是否參與了ABD的發(fā)病，是否在ABD發(fā)病中輔助B細(xì)胞產(chǎn)生功能性抗體引起一系列免疫損傷，以期對(duì)ABD的發(fā)病機(jī)制進(jìn)行進(jìn)一步探索，并為ABD的治療提供新的思路。目的：探索Tfh細(xì)胞與天皰瘡和大皰性類天皰瘡為代表的自身免疫性皰病的相關(guān)性。方法：收集天皰瘡和大皰性類天皰瘡患者及健康對(duì)照者外周血，提取外周血單個(gè)核細(xì)胞（PBMC）并分離血清，通過流式細(xì)胞術(shù)檢測(cè)患者和正常對(duì)照者外周血中Tfh細(xì)胞水平，通過酶聯(lián)免疫吸附試驗(yàn)（ELISA）檢測(cè)患者和正常對(duì)照者血清中IL-21水平，分析Tfh和IL-21在患者與正常對(duì)照者中的差異以及與疾病嚴(yán)重程度的關(guān)系，分析Tfh與ABD發(fā)病的相關(guān)性。結(jié)果：大皰性類天皰瘡患者外周血中Tfh水平高于健康對(duì)照者（中位數(shù)分別為11.25%和4.95%, P<0.001）；Tfh水平與反映病情嚴(yán)重程度的抗BP180-NC16A抗體滴度呈正相關(guān)（R=0.712, P<0.01）；大皰性類天皰瘡患者血清中IL-21水平高于健康對(duì)照組（中位數(shù)分別為103.98pg/ml和46.77pg/ml,P<0.001），且IL-21水平與其血清中抗BP180-NC16A滴度呈正相關(guān)（R=0.578,P=0.030）；治療后，隨著患者病情的緩解及抗體滴度的下降，其外周血中Tfh水平較治療前下降（中位數(shù)分別為10.50%和4.10%, P=0.003），治療后血清中IL-21水平也較治療前下降（中位數(shù)分別為99.98pg/ml和64.08pg/ml,P=0.012）。天皰瘡患者外周血中Tfh水平高于健康對(duì)照組（中位數(shù)分別為10.00%和4.80%, P=0.001），血清中IL-21水平也高于正常對(duì)照組（中位數(shù)分別為72.84pg/ml和60.34pg/ml, P=0.011）；治療后，隨著患者病情的緩解，其外周血中Tfh細(xì)胞比例較治療前下降（中位數(shù)分別為9.90%和5.00%，P=0.018），其治療后IL-21水平較也治療前下降（中位數(shù)分別為84.46pg/ml和69.58pg/ml,P=0.028）。結(jié)論：以天皰瘡和大皰性類天皰瘡為代表的ABD患者外周血Tfh細(xì)胞及其分泌的主要細(xì)胞因子IL-21水平均明顯升高，，且BP患者中Tfh及IL-21水平均與反映病情嚴(yán)重程度的特異性抗體滴度呈正相關(guān)；治療后，隨著疾病緩解，患者外周血中Tfh及IL-21水平均顯示出較治療前下降趨勢(shì)。以上結(jié)果提示Tfh可能在ABD發(fā)病過程中起著重要作用，且與疾病嚴(yán)重程度及活動(dòng)度相關(guān)，通過本實(shí)驗(yàn)，為進(jìn)一步探索ABD中自身抗體的來源提供了新的線索，進(jìn)一步完善了ABD發(fā)病機(jī)制，并為ABD的治療提供新的思路。

    Autoimmune bullous diseases (ABD) are a group of classic autoimmunediseases characterized by clinical erythema, blister, bulla and erosion. Commonlyseen in elderly individuals, they are famous of intractable and being tend torecurrent attacks. ABD is generally classified in two broad categories:intraepidermal and subepidermal bullous diseases. Pemphigus is most common inthe former group, and bullous pemphigoid is the typical representation of the latergroup[1].The immunological pathogenesis of ABD mediated by autoantibodies isgenerally accepted. It is confirmed that the binding of autoantibody to antigeninitiates a series of immune inflammatory events including activation ofcomplement, accumulation of inflammatory cell, release of proteinase andlysosomal enzyme that disrupts the critical adhesion molecules of epidermis orbasement membrane zone or affects the function of adhesion molecules directly,then leads to clinical blister or bulla[2].Recently advances were showed about thepathogenesis of ABD. It is clear that Dsg3and Dsg1are the major antigens ofpemphigus vulgaris and pemphigus foliaceus respectively and BPAG1(BP230)and BPAG2(BP180) are the antigen of bullous pemphigoid[3], but the source and emerging mechanism of autoantibody is not clear yet.Previous studies have shown that CD4+Th cells are required for B cellsprimed by T cell-denpendent antigen during the process of antibody production.Antibody secreting cells produced in germinal center (GC) where somatichypermutation, class switching of Ig and the choice of high affinity occurred. Thcells play a critical role in formation of GC, choice of high affinity B cellsdifferentiate into memory cells or plasma cells and maintain a prolonged humoralimmune response[4]. From recent studies it is believed that T follicular helpercells (Tfh) are responsible for helping B cells during antibody response. Tfhlocalize to the follicles, the transcription factor of them is Bcl-6, Tfh cellsexpressed high levels of CXCR5, ICOS, CD40L, OX40, PD-1, SAP, and so on,the combination of them with corresponding receptors or ligands on B cellssurface ensures Tfh help B cells produce high affinity antibodies. Tfh cellsproduce high levels of IL-21, by which they regulate the GC reactionappropriately[5].Recent studies support the involvement of Tfh cells in autoimmune diseases.Abnormal levels of Tfh cells were observed participating in GC formation andthe production of autoantibodies in many mice or patients with autoimmunediseases, including SLE, RA, MG and so on. Blocking the function of importantmolecules such as ICOS, CD40L and IL-21in Tfh resulted in reduced productionof antibodies[6-10]. Due to the source and mechanism of autoantibodies with ABDis not yet clear, and the major function of Tfh cells is help B cells to produceantibodies, by detecting the populations of Tfh cells in peripheral blood ofpatients with pemphigus and bullous pemphigoid, we explore preliminarywhether Tfh is involved in the incidence of ABD, seek further complement in thepathogenesis of ABD and find new ideas for the therapy of ABD. Objective: To explore the correlation of Tfh cells to ABD which representedby pemphigus and bullous pemphigoid.Methods: Peripheral blood was collected from pemphigus and BP patientsand normal controls. PBMC was extracted and serum was separated, doubleantibody sandwich ELISA was performed to measure the levels of serum IL-21,flow cytometry was performed to detect the level of Tfh in PBMCs, by which weexplore the correlation of Tfh cells to ABD.Results: The increase of Tfh cells in the peripheral blood of BP patients wasobserved compared with healthy controls (median11.25%versus4.95%,P<0.001). The populations of Tfh cells were positively correlated to the titer ofanti-BP180-NC16A in BP patients (R=0.712, P<0.01). Compared with healthycontrols, serum level of interleukin21(IL-21) was higher in the peripheral bloodof BP patients (103.98pg/ml versus46.77pg/ml, P<0.001). The levels of IL-21were positively correlated to the titer of anti-BP180-NC16A in BP patients(R=0.578, P=0.030). After therapy, accompanied by the remission of diseasesand decrease of titers of autoantibodies, the populations of Tfh cells weredescended compared with themselves before therapy (median10.50%versus4.10%, P=0.003). And the IL-21levels were declined too (median99.98pg/mlversus64.08pg/ml, P=0.012).Similarly, The increase of Tfh cells in the peripheral blood of pemphiguspatients was observed compared with healthy controls (median10.00%versus4.80%, P=0.001). Compared with healthy controls, serum level of IL-21washigher in the peripheral blood of pemphigus patients (median72.84pg/ml versus60.34pg/ml, P=0.011). After therapy, accompanied by the remission of diseasesand decrease of titers of autoantibodies, the populations of Tfh cells weredescended compared with themselves before therapy (median9.90%versus 5.00%，P=0.018); The IL-21levels were declined compared with themselvesbefore therapy too (median84.46pg/ml versus69.58pg/ml, P=0.028).Conclusion: Significant increase of Tfh cells populations were observed inpatients with pemphigus and BP, and IL-21, the major cytokine secteted by Tfhwas also increased compared with normal controls. Especially, Both of the Tfhpopulations and the IL-21levels show positive correlations with the titer ofanti-BP18NC16A autoantibodies in BP patients. After therapy, with the relief ofthe diseases, both of the levels of Tfh and IL-21were decreased compared withthemselves before therapy. These results suggest that Tfh cells play a critical rolein the autoantibody production of ABD and positively correlated with the diseaseseverity and activity. Our results provide new cues for searching of the source ofautoantibodies in ABD, add more mechanisms of ABD, and supply new ideasabout the therapy of ABD.

          濾泡輔助性T細(xì)胞（Tfh）與自身免疫性皰病的相關(guān)性研究

縮略語表5-7中文摘要7-10Abstract10-13前言14-16文獻(xiàn)回顧16-41    一、自身免疫性皰病16-32    二、濾泡輔助性 T 細(xì)胞（Tfh）32-37    三、Tfh 細(xì)胞與自身免疫性疾病的關(guān)系37-411 材料41-432 方法43-463 結(jié)果46-574 討論57-62小結(jié)62-63參考文獻(xiàn)63-75個(gè)人簡(jiǎn)歷和研究成果75-76致謝76

  本文關(guān)鍵詞：濾泡輔助性T細(xì)胞（Tfh）與自身免疫性皰病的相關(guān)性研究，由筆耕文化傳播整理發(fā)布。