本文選題：類天皰瘡　切入點(diǎn)：大皰性　出處：《第四軍醫(yī)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：大皰性類天皰瘡(bullous pemphigoid,BP)是一種好發(fā)于老年人,臨床表現(xiàn)為緊張性水皰、大皰的慢性自身免疫性水皰性皮膚病,治療目前主要依靠應(yīng)用糖皮質(zhì)激素和免疫抑制劑等方法。BP病情易反復(fù)發(fā)作,長(zhǎng)期應(yīng)用激素等治療產(chǎn)生的副作用是臨床實(shí)際工作中急需解決的問題[1]。 BP組織病理學(xué)表現(xiàn)為表皮下水皰形成,皰內(nèi)可見到以嗜酸性粒細(xì)胞為主,包括淋巴細(xì)胞及中性粒細(xì)胞等多種炎細(xì)胞的浸潤(rùn)�；颊哐逯锌梢詸z測(cè)到特異性針對(duì)皮膚真表皮連接處的基底膜帶(BMZ)的自身抗體,直接免疫熒光亦可以發(fā)現(xiàn)患者皮膚BMZ有抗體(IgG、IgE等)和補(bǔ)體沉積。目前研究認(rèn)為BP的發(fā)病機(jī)制是由患者體內(nèi)循環(huán)自身抗體介導(dǎo)補(bǔ)體激活、肥大細(xì)胞及中性粒細(xì)胞等炎細(xì)胞參與而最終導(dǎo)致皮膚真表皮交界處水皰發(fā)生的復(fù)雜過程[2]。 BP180屬于膠原蛋白家族成員,位于皮膚基底膜帶半橋粒上的一種Ⅱ型跨膜糖蛋白,又稱ⅩⅦ型膠原(COL17)或大皰性類天皰瘡抗原2 (BPAg2),在真表皮連接中發(fā)揮重要作用。目前認(rèn)為BP180為BP致病性自身抗體所識(shí)別的主要靶抗原,其細(xì)胞外區(qū)域第16非膠原編碼區(qū)(NC16A)是致病性自身抗體所識(shí)別的主要靶表位區(qū)[3]。BP致病性自身抗體與BP180等自身抗原特異性結(jié)合并激活補(bǔ)體,造成明顯的免疫炎癥反應(yīng),導(dǎo)致真表皮分離最終發(fā)生表皮下水皰。Nishie[4]等在BP180人源化動(dòng)物模型上證實(shí)BP患者血清自身抗體在BP發(fā)病中發(fā)揮了關(guān)鍵作用,更深層次地揭示了BP的發(fā)病機(jī)理。在此過程中,自身抗體與BP180等自身抗原結(jié)合,進(jìn)而激活補(bǔ)體是非常重要的環(huán)節(jié)。因此,我們通過制備人源性基因工程單鏈抗體,利用其能特異性識(shí)別靶抗原而又因不含有補(bǔ)體結(jié)合位點(diǎn)的Fc段,不能激活補(bǔ)體的特性,封閉與BP自身抗體結(jié)合的自身抗原表位,阻斷自身抗體與自身抗原的結(jié)合,干預(yù)后續(xù)的免疫炎癥反應(yīng),從而消除自身抗體的致病作用,探尋一種新的治療BP的生物學(xué)方法。 我們前期構(gòu)建了針對(duì)BP180-NC16A的單鏈抗體(single chain Fv,scFv),并實(shí)現(xiàn)了其可溶性表達(dá)及純化[5, 6]。本研究對(duì)所獲得的單鏈抗體進(jìn)行功能學(xué)的鑒定,觀察其對(duì)BP血清IgG自身抗體的競(jìng)爭(zhēng)性抑制作用,構(gòu)建BP體外冰凍切片模型,并在此模型中驗(yàn)證所獲得的單鏈抗體可競(jìng)爭(zhēng)性抑制由于BP自身抗體結(jié)合抗原引起后續(xù)免疫炎癥反應(yīng),從而導(dǎo)致真表皮分離的發(fā)病過程。我們的研究為BP特異性的生物治療提供實(shí)驗(yàn)依據(jù),同時(shí)也為以自身抗體作為主要致病機(jī)制的自身免疫病治療提供了一個(gè)新的研究方向。 目的:對(duì)人源性抗BP180單鏈抗體進(jìn)行生物學(xué)功能的鑒定。 方法:蛋白親和層析的方法純化BP血清自身抗體,通過競(jìng)爭(zhēng)性ELISA、競(jìng)爭(zhēng)性免疫熒光和補(bǔ)體活化的競(jìng)爭(zhēng)性抑制實(shí)驗(yàn)來(lái)觀察所制備的單鏈抗體對(duì)自身抗體的競(jìng)爭(zhēng)抑制作用。構(gòu)建BP體外冰凍切片模型,利用此模型觀察所制備的單鏈抗體對(duì)自身抗體所致真表皮分離的抑制作用。 結(jié)果:抗BP180-NC16A scFv對(duì)BP血清IgG抗體具有較好的抑制效果。競(jìng)爭(zhēng)性ELISA各濃度組間A450值下降明顯,在0-60μg范圍內(nèi)成劑量依賴關(guān)系,最大抑制率可達(dá)到69.50%(與對(duì)照組相比均有統(tǒng)計(jì)學(xué)意義,P0.01);間接免疫熒光實(shí)驗(yàn)及補(bǔ)體活化的競(jìng)爭(zhēng)抑制實(shí)驗(yàn)在抑制前可見線性熒光沉積條帶,經(jīng)scFv抑制后則無(wú)熒光條帶。BP冰凍切片組織模型HE染色可見真表皮的分離,加入不同濃度的單鏈抗體后真表皮的分離受到明顯的抑制。 結(jié)論:我們所制備的抗大皰性類天皰瘡抗原BP180單鏈抗體對(duì)致病性自身抗體結(jié)合抗原具有一定的競(jìng)爭(zhēng)抑制作用,為工程抗體封閉自身抗體抗原表位這一治療策略提供了實(shí)驗(yàn)依據(jù)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R758.66

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相關(guān)期刊論文 前3條

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