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Application of up-to date immunoelectron microscopy for the study of pathogenesis of skin blistering disease.

应用最新免疫电子显微镜研究皮肤水疱病的发病机制。

基本信息

批准号：
03454275
负责人：
NISHIKAWA Takeji
金额：
$ 4.16万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

Many different types of immuno EM techniques have been introduced in dermatological research varing from relatively simple ones which could be used in most laboratories ( pre-embedding peroxidase method) to complex procedure relevant mainly to the specialized research laboratory (post-embedding, cryofixation and cryosubstitution technique). In this project, we have employed up-to date low temperature immunoelectron microscopic techniques for the study of skin blistering disease including bullous pemphigoid antigen and epidermolysis bullosa acquisita antigen. Previous immuno-peroxidase method could demonstrate the ultrastructural localization of the immunoreactants only fairly accurately in relation to the subcomponents of the dermo-epidermal junction. Because the size of peroxidase-diaminobenzidine reaction products is large and prone to diffuse, and inevitably obscure the underlying structure. Applying cryofixation and cryosubstitution techniques without using chemical fixatives in co … More njunction with colloidal gold, we have demonstrated that epidermolysis bullosa acquisita antigen, a target molecule of the autoantibodies of the patient, localized to both dermal and lamina densa ends of anchoring fibrils, but not to the central banded portion itself. Anchoring fibrils is a structure of 785nm in length. Colloidal gold but not peroxidase can elucidate the fine ultrastructural localization of the target epitope on such small structure.Bullous pemphigoid antigen is composed of two major antigenic proteins of the 230-kD antigen and the 180-kD antigen. We have used this new immunoelectron microscopic technique to localize the epitopes for autoantibodies against each antigen. Autoantibodies against each antigen were affinity-purified using nitrocellulose membrane, which was blotted with SDS-PAGE fractionated antigens from human epidermal extract as the immunoabsorbent. Postembedding immunogold electron microscopy was performed using low temperature techniques. The 230-kD antigen only localized to the intracellular domain of hemidesmosome. In contrast, the autoantibodies against the 180-kD antigen localized along the plasma membrane of hemidesmosome. These result suggest that the autoantibodies against the 180 and 230-kD bullous pemphigoid antigen may play different roles in the blister formation in patients with bullous pemphigoid. Less

许多不同类型的免疫电镜技术已被引入皮肤病学研究中，从可在大多数实验室使用的相对简单的技术（包埋前过氧化物酶方法）到主要与专业研究实验室相关的复杂程序（包埋后、冷冻固定和冷冻替代技术）。在这个项目中，我们采用了最新的低温免疫电子显微镜技术来研究皮肤起泡疾病，包括大疱性类天疱疮抗原和大疱性表皮松解抗原。以前的免疫过氧化物酶方法只能相当准确地证明免疫反应物的超微结构定位与真皮-表皮连接的亚成分的关系。因为过氧化物酶-二氨基联苯胺反应产物的尺寸很大并且容易扩散，并且不可避免地掩盖了底层结构。应用冷冻固定和冷冻替代技术而不使用化学固定剂与胶体金结合，我们证明了获得性大疱性表皮松解抗原（患者自身抗体的目标分子）定位于锚定原纤维的真皮和致密层末端，但不定位于中央带状部分本身。锚定原纤维是一种长度为 785nm 的结构。胶体金而非过氧化物酶可以阐明目标表位在如此小的结构上的精细超微结构定位。大疱性类天疱疮抗原由230-kD抗原和180-kD抗原两种主要抗原蛋白组成。我们使用这种新的免疫电子显微镜技术来定位针对每种抗原的自身抗体的表位。针对每种抗原的自身抗体使用硝酸纤维素膜进行亲和纯化，并用来自人表皮提取物的 SDS-PAGE 分级抗原作为免疫吸收剂进行印迹。使用低温技术进行包埋后免疫金电子显微镜检查。 230-kD 抗原仅定位于半桥粒的细胞内结构域。相反，针对 180-kD 抗原的自身抗体沿着半桥粒的质膜定位。这些结果表明，针对180和230-kD大疱性类天疱疮抗原的自身抗体可能在大疱性类天疱疮患者水疱形成中发挥不同的作用。较少的