人源性抗大疱性类天疱疮抗原BP180单链抗体的生物学功能研究

发布时间：2018-03-16 21:07

本文选题：类天疱疮　切入点：大疱性　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：大疱性类天疱疮(bullous pemphigoid,BP)是一种好发于老年人,临床表现为紧张性水疱、大疱的慢性自身免疫性水疱性皮肤病,治疗目前主要依靠应用糖皮质激素和免疫抑制剂等方法。BP病情易反复发作,长期应用激素等治疗产生的副作用是临床实际工作中急需解决的问题[1]。 BP组织病理学表现为表皮下水疱形成,疱内可见到以嗜酸性粒细胞为主,包括淋巴细胞及中性粒细胞等多种炎细胞的浸润。患者血清中可以检测到特异性针对皮肤真表皮连接处的基底膜带(BMZ)的自身抗体,直接免疫荧光亦可以发现患者皮肤BMZ有抗体(IgG、IgE等)和补体沉积。目前研究认为BP的发病机制是由患者体内循环自身抗体介导补体激活、肥大细胞及中性粒细胞等炎细胞参与而最终导致皮肤真表皮交界处水疱发生的复杂过程[2]。 BP180属于胶原蛋白家族成员,位于皮肤基底膜带半桥粒上的一种Ⅱ型跨膜糖蛋白,又称ⅩⅦ型胶原(COL17)或大疱性类天疱疮抗原2 (BPAg2),在真表皮连接中发挥重要作用。目前认为BP180为BP致病性自身抗体所识别的主要靶抗原,其细胞外区域第16非胶原编码区(NC16A)是致病性自身抗体所识别的主要靶表位区[3]。BP致病性自身抗体与BP180等自身抗原特异性结合并激活补体,造成明显的免疫炎症反应,导致真表皮分离最终发生表皮下水疱。Nishie[4]等在BP180人源化动物模型上证实BP患者血清自身抗体在BP发病中发挥了关键作用,更深层次地揭示了BP的发病机理。在此过程中,自身抗体与BP180等自身抗原结合,进而激活补体是非常重要的环节。因此,我们通过制备人源性基因工程单链抗体,利用其能特异性识别靶抗原而又因不含有补体结合位点的Fc段,不能激活补体的特性,封闭与BP自身抗体结合的自身抗原表位,阻断自身抗体与自身抗原的结合,干预后续的免疫炎症反应,从而消除自身抗体的致病作用,探寻一种新的治疗BP的生物学方法。我们前期构建了针对BP180-NC16A的单链抗体(single chain Fv,scFv),并实现了其可溶性表达及纯化[5, 6]。本研究对所获得的单链抗体进行功能学的鉴定,观察其对BP血清IgG自身抗体的竞争性抑制作用,构建BP体外冰冻切片模型,并在此模型中验证所获得的单链抗体可竞争性抑制由于BP自身抗体结合抗原引起后续免疫炎症反应,从而导致真表皮分离的发病过程。我们的研究为BP特异性的生物治疗提供实验依据,同时也为以自身抗体作为主要致病机制的自身免疫病治疗提供了一个新的研究方向。目的:对人源性抗BP180单链抗体进行生物学功能的鉴定。方法:蛋白亲和层析的方法纯化BP血清自身抗体,通过竞争性ELISA、竞争性免疫荧光和补体活化的竞争性抑制实验来观察所制备的单链抗体对自身抗体的竞争抑制作用。构建BP体外冰冻切片模型,利用此模型观察所制备的单链抗体对自身抗体所致真表皮分离的抑制作用。结果:抗BP180-NC16A scFv对BP血清IgG抗体具有较好的抑制效果。竞争性ELISA各浓度组间A450值下降明显,在0-60μg范围内成剂量依赖关系,最大抑制率可达到69.50%(与对照组相比均有统计学意义,P0.01);间接免疫荧光实验及补体活化的竞争抑制实验在抑制前可见线性荧光沉积条带,经scFv抑制后则无荧光条带。BP冰冻切片组织模型HE染色可见真表皮的分离,加入不同浓度的单链抗体后真表皮的分离受到明显的抑制。结论:我们所制备的抗大疱性类天疱疮抗原BP180单链抗体对致病性自身抗体结合抗原具有一定的竞争抑制作用,为工程抗体封闭自身抗体抗原表位这一治疗策略提供了实验依据。
[Abstract]:Bullous pemphigoid (bullous pemphigoid BP) is a kind of good in the elderly, the clinical manifestations of tension blisters, bullous chronic autoimmune blistering skin disease treatment, mainly rely on the use of corticosteroids and immunosuppressive agents such as.BP was easy to repeated attacks, side effects of long-term use of hormone the treatment is in urgent need of clinical practical work to solve the problem of [1].
BP histopathology revealed subepidermal blister formation, blister can be seen in eosinophil infiltration, including a variety of lymphocytes and neutrophils. Serum was detected in the basement membrane specific to the dermal epidermal junction zone (BMZ) antibodies, direct immunofluorescence also can be found in patients with cutaneous BMZ antibodies (IgG, IgE) and complement deposition. The current research that the pathogenesis of BP by autoantibodies mediated complement activation, mast cells and neutrophils participate and lead eventually to the dermal epidermal junction [2]. complex process at the blister
BP180 belongs to the family of collagen, a type II is located in the basement membrane hemidesmosomes on transmembrane glycoprotein, also called collagen (COL17) or bullous pemphigoid antigen 2 (BPAg2), play an important role in the true skin connection. Now that the main target antigen recognized by BP180 BP disease autoantibodies and its extracellular domain sixteenth non collagen encoding region (NC16A) is the pathogenic autoantibodies identified major target epitope [3].BP pathogenic autoantibodies and BP180 autoantigen specific binding and complement activation, resulting in immune inflammatory reaction obviously, leading to true epidermal separation occurs eventually subepidermal blister.Nishie[4] in BP180 humanized animal model confirmed BP patients with serum autoantibodies in the pathogenesis of BP play a key role to further reveal the pathogenic mechanism of BP. In this process, autoantibodies and BP180 itself Antigen binding and complement activation is very important. Therefore, we prepare humanized gene engineering antibody through the system, the specific recognition of target antigens and for not containing Fc segment complement binding sites, complement activation properties not closed self epitopes with BP antibody. Blocking the binding of autoantibodies and antigens, the immune inflammatory response following intervention, thereby eliminating the pathogenic role of autoantibodies, explore a new biological method for the treatment of BP.
We previously constructed the scFv BP180-NC16A (single chain Fv, scFv), and realized its soluble expression and purification of [5 6]., this study identified functional of scFv obtained, to observe the serum BP autoantibodies IgG competitive inhibition, construction of frozen sections of BP in vitro model, single strand verify the obtained antibody and in this model the competitive inhibition due to BP antibody binding antigen caused by subsequent inflammatory reaction, which leads to the pathogenesis of true epidermal separation. Our study provides an experimental basis for BP specific biological treatment, but also provides a new research direction for the treatment of autoimmune diseases the autoantibodies as the main pathogenic mechanism.
Objective: to identify the biological function of human anti BP180 single chain antibody.
Methods: serum autoantibodies purified BP protein affinity chromatography method, through competitive ELISA, competitive immunofluorescence and complement activation of the competitive inhibition experiment to observe the preparation of single chain antibody inhibition on autoantibody competition. To construct BP frozen in vitro model, inhibition was observed by using the model of single chain antibody preparation the separation of true skin caused by autoantibodies.
Results: the anti BP180-NC16A scFv has good inhibitory effect on serum BP IgG antibody. Competitive ELISA among the A450 groups decreased significantly, with a dose-dependent in the 0-60 g range, the maximum inhibition rate can reach 69.50% (compared with the control group had statistical significance, P0.01); indirect immunofluorescence and complement activation the competitive inhibition experiment in inhibiting the visible deposition of linear fluorescent bands by scFv after inhibition of no fluorescence bands.BP frozen tissue model HE staining visible true epidermal separation, single chain antibody with different concentrations after the dermal epidermal separation was inhibited.
Conclusion: our anti bullous pemphigus antigen BP180 scFv has a competitive inhibitory effect on the pathogenic autoantibody binding antigen, which provides an experimental basis for the engineering antibody to block autoantibody epitopes.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.66

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