The identification of autoantigens in mucous membrane pemphigoid using immortalized oral mucosal keratinocytes.
The identification of autoantigens in mucous membrane pemphigoid using immortalized oral mucosal keratinocytes.
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使用永生化口腔粘膜角质形成细胞鉴定粘膜类天疱疮中的自身抗原。
DOI：
10.1111/jop.12780
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Kitagawa Y.
中科院分区：
文献类型：
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作者：
Kamaguchi M;Iwata H;Miyauchi T;Ujiie H;Ujiie I;Nomura T;Ohga N;Shimizu H;Kitagawa Y.
粘膜类天疱疮（Mucous membrane pemphigoid，MMP）是一种罕见的慢性自身免疫性上皮下水疱性疾病，靶向于包括胶原XVII（collagen XVII，COL 17）在内的多种基底膜区（BMZ）蛋白。MMP的循环自身抗体由于其较低的滴度而常常未被检测到。口腔粘膜是检测MMP患者自身抗体的有价值的基质。然而，获得正常人口腔粘膜比获得正常人皮肤更困难。我们建立了永生化的正常人口腔粘膜角质形成细胞（OMK），并进行免疫印迹使用永生化的OMK裂解液检测自身抗原MMP.MethodsImmortalized OMK产生的原OMK使用HPV的E6/E7蛋白。我们比较了原代OMK和永生化OMK之间主要BMZ蛋白的蛋白表达水平。我们进行免疫印迹检测自身抗原使用细胞裂解物从永生化OMK在30 MMP patients.ResultsThere原OMK和永生化OMK的BMZ蛋白的蛋白质表达水平方面没有显着差异，包括COL 17，层粘连蛋白332，整合素α6/β4，胶原蛋白VII，胶原蛋白IV。永生化OMK的细胞裂解物有效地识别了60%（18/30）的MMP血清中的MMP自身抗原。我们发现了一个有趣的MMP自身抗体的情况下，优先反应的120-kD蛋白，是一个胞外结构域的COL 17。结论我们证明了永生化OMK细胞裂解液是一个可靠的基质检测MMP自身抗原。这种新开发的免疫印迹分析方法有望有助于MMP的诊断。
BackgroundMucous membrane pemphigoid (MMP) is a rare chronic autoimmune subepithelial blistering disorder, targeting multiple basement membrane zone (BMZ) proteins including collagen XVII (COL17). Circulating autoantibodies of MMP are often undetected due to their lower titers. The oral mucosa is a valuable substrate for the detection of autoantibodies in MMP patients. However, obtaining normal human oral mucosa is more difficult than obtaining normal human skin. We established immortalized normal human oral mucosal keratinocytes (OMKs) and performed immunoblotting using immortalized OMK lysate for detecting autoantigens in MMP.MethodsImmortalized OMKs were generated from primary OMKs using E6/E7 proteins of HPV. We compared the protein expression levels of major BMZ proteins between primary OMKs and immortalized OMKs. We performed immunoblotting to detect autoantigens using cell lysates from immortalized OMKs in 30 MMP patients.ResultsThere were no significant differences between primary OMKs and immortalized OMKs in terms of protein expression levels of the BMZ proteins, including COL17, laminin 332, integrin α6/β4, collagen VII, and collagen IV. Cell lysates of immortalized OMKs effectively identified MMP autoantigens in 60% (18/30) of MMP sera. We found an interesting case of MMP whose autoantibodies preferentially reacted to the 120‐kD protein that is an ectodomain of COL17.ConclusionWe demonstrated that a cell lysate of immortalized OMKs is a reliable substrate for the detection of MMP autoantigens. This newly developed immunoblotting analysis method promises to contribute to the diagnosis of MMP.