粒细胞相关的自身抗体引起的皮肤起泡
Summary
在动物模型中描述在我们目前的工作中，纯化的IgG抗体对拉伸的200个氨基酸（氨基酸757-967）的胶原纤维VII反复被注入到小鼠体内，再现起泡表型以及直方和免疫病理学特征对人体acquisita大疱性表皮松解症（EBA）<sup> 1</sup>。
Abstract
自身免疫性现象发生在健康人，但，自我宽容失败时，可能会导致自体免疫反应在具体的病理。据Witebsky的假设，诊断为自身免疫性疾病中的标准之一是在实验动物中的疾病的再现由被动转移的自身抗体。大疱性表皮松解症acquisita（EBA），原型器官特异性自身免疫性疾病，皮肤和粘膜，一些实验模型，最近成立的。在动物模型中描述在我们目前的工作中，纯化的IgG抗体对拉伸的200个氨基酸（氨基酸757-967）的胶原纤维VII反复被注入到小鼠体内，再现起泡表型以及直方和免疫病理学特征对人体EBA 1。全面爆发普遍的疾病，通常第一次注射后5-6天，疾病的程度与剂量的管理Çollagen的VII-特异性IgG。在实验EBA组织损伤（水疱形成）根据粒细胞的募集和活化组织结合自身抗体-2，-4。因此，该模型允许粒细胞依赖的炎性途径参与自身免疫性组织损伤的解剖，作为模型只再现传出神经的自体免疫反应的T细胞独立的相位。此外，它的值被强调的一些研究表明自身抗体在体内的泡罩诱发电位和调查-6 EBA 1,3，水疱形成的机制。最后，这种模式将极大地促进新的自身抗体引起的疾病的消炎疗法的发展。总体而言，EBA被动转移动物模型的可访问性和指导性的疾病模型，将有助于研究人员不仅分析的的EBA发病，但回答基本的生物学和临床重要的自身免疫问题。
Protocol
1。致病抗体的制备注意：该抗体的纯化和浓度应在同一天进行，作为抗体是不是要被存储在0.1M甘氨酸pH为2.5-3（洗脱缓冲液）过夜（ON）。 亲和纯化的IgG：使用免疫兔血清25毫升： 解冻后的兔血清对在4°C，混合，以1:1 1xPBS（运行缓冲液），并在1260 XG离心10分钟，在20°C。 ，用滤纸过滤步骤可能包括离心后的情况下，血清脂肪。 同时，洗G矩…
Representative Results
的被动转移抗原特异性抗体，结果在一个完全成熟的病鼠在临床，病理和免疫病理的水平，类似的人EBA。水疱，痂皮，糜烂和脱发发展的耳朵，猪嘴，爪，腿，背部和眼睛周围的老鼠。第一临床疾病的征兆，很可能会出现在耳朵上和/或头部区域。如果在中冰冻切片的病灶周围皮肤的真皮表皮交界处，检测到特异性的兔IgG和鼠补体C3沉积直接。在皮损部位表皮下疱和炎症浸润的组织学。 <p class="j…
Discussion
被动转移到实验动物的自身抗体是一个重要的方法展示他们的致病性，-12。通过这种方法获得的动物模型，除了是自身免疫13的间接证据，允许传出的调查阶段的致病机理。抗体诱导的粒细胞依赖的皮肤起泡性大疱性表皮松解症acquisita（EBA）的被动转移模型被用于解剖组织损伤的机制，在自身免疫性皮肤病。它的出现，作为一个精美的启发性疾病模型研究的根本，远远超出对胶原?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者承认，从德意志研究联合会SI-1281/4-1的和BIOSS从弗赖堡大学医学系（CS）的补助金。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| recombinant antigen | The plasmids encoding for the recombinant forms of murine collagen VII are available from the corresponding author. | ||
| immune rabbit serum | www.eurogentec.com | We had New Zealand White rabbits immunized with 200 μg of antigen, 3 times at 2 week intervals. For this purpose we have used the services of Eurogentec S.A., Belgium. | |
| Protein G agarose | Roche Applied Science | 11243233001 | |
| Balb/c mice | Charles River Laboratories | ||
| OCT compound, Tissue-Tek | Sakura Finetek | 4583 | OCT, optimal cutting temperature |
| Cryomold standard | Sakura Finetek | 4557 | 25 mm × 20 mm × 5 mm |
| Cryomold intermediate | Sakura Finetek | 4566 | 15 mm × 15 mm × 5 mm |
| Uni-Link-Einbettkassette | R. Langenbrinck | 09-0503 | Histology processing and embedding cassettes |
| Spezial-Tatowierfarbe Schwarz | H. Hauptnerund & Richard Herberholz GmbH & Co. KG | 71492000 | tattooing paste |
| Tatowierzange TZ1 | EBECO E. Becker & Co GmbH | tattooing device | |
| Heparin | Carl Roth GmbH & Co. | 7692.1 | |
| Formaldehyde 37% | Carl Roth GmbH & Co. | 7386 | |
| Ketamine hydrochloride | Sigma-Aldrich Chemie GmbH | K2753-1G | |
| Xylazine hydrochloride | Sigma-Aldrich Chemie GmbH | X1251-1G | |
| sterile PBS | Biochrom | L182-50 | |
| digital camera | Nikon | Coolpix 5400 | |
| Syringe driven filter unit 0.45 μm | Millipore | ||
| Caliper | Mitutoyo 7309 | 1667338 | Farnell (distributor) |
| disease scoring sheet | example enclosed |
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Tags
Granulocyte-dependentAutoantibody-inducedSkin BlisteringAutoimmune PhenomenaSelf-toleranceAutoimmune ResponseWitebsky’s PostulatesDiagnosing Autoimmune DiseasePassive Transfer Of AutoantibodiesEpidermolysis Bullosa Acquisita (EBA)Experimental ModelsCollagen VIIBlistering PhenotypeHisto- And Immunopathological FeaturesHuman EBAWidespread DiseaseDose Of Administered Collagen VII-specific IgGGranulocyte Recruitment And ActivationInflammatory Pathway
Cite This Article
Csorba, K., Sitaru, S., Sitaru, C. Granulocyte-dependent Autoantibody-induced Skin Blistering. J. Vis. Exp. (68), e4250, doi:10.3791/4250 (2012).