CHARACTERIZATION OF SUB-EPITHELIAL ANTIGENS

上皮下抗原的表征

• 批准号: 3157344
• 负责人: ROBERT E BURGESON
• 金额: $ 9.65万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-23 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3157344
• 关键词: affinity chromatography; antibody; basement membrane; collagen; complementary DNA; electron microscopy; epidermolysis bullosa; epithelium; extracellular matrix; gel electrophoresis; heterozygote; histochemistry /cytochemistry; human tissue; immunochemistry; intercellular connection; keratin; keratinocyte; laboratory mouse; laboratory rabbit; laminin; monoclonal antibody; protein sequence; skin; surface antigens; tissue /cell culture

Human skin keratinocytes are affixed to the underlying dermal matrix by a complex attachment mechanism, many elements of which are unique to external eipthelia. These include the hemidesmosomes which mediate attachment of keratin filaments to the basolateral plasma membrane, anchoring filaments that appear to bridge the hemidesmosomes to the lamina densa of the basement membrane, and the anchoring fibril network, that attaches the lamina densa to the subjacent dermis by physical entrapment of dermal fibrous elements. Failure of this attachment complex results in several forms of the blistering diseases jointly termed Epidermolysis Bullosa (EB). While the phenomena of dermal-epidermal separation is well documented, the molecular events leading of these separations are not unambiguously known. During the past three years of funding, this grant has supported studies that identified a large and complex C-terminal globular domain of type VII collagen (NC-1) and indicated involvement of this domain in a new model of the anchoring fibril network. The model is based upon structural characterization of type VII collagen and ultrastructural immunolocalization of NC-1. The model predicts interactions of NC-1 with itself, with type IV collagen and with other intrinsic basement membrane components both with the lamina densa and within structures we have termed "anchoring plaques". This application proposes continuation of these studies to: (1) investigate the possible interactions of NC-1 with itself and with domains of type IV collagen and other basement membrane proteins using solid phase interaction assays and biophysical techniques; (2) to attempt to isolate additional NC-1 binding proteins by affinity chromatography and to characterize their structures; (3) to begin identification of the structural components of hemidesmosomes by first producing monoclonal antibodies to membrane fractions enriched for them and use of these antibodies for immunoisolation and structural characterization of them; and (4) to assess the ability of cultured EB keratinocytes to synthesize type VII collagen using cDNA hybridization and immunoblotting techniques, and to evaluate the defective anchoring fibril networks of individuals with recessive dystrophic EB relative to the above described model, using electron microscope immunohistochemistry.

人类皮肤角质形成细胞附着在真皮下面 矩阵由复杂的附着机制组成，许多元素 这是外部上皮所特有的。 这些包括 介导角蛋白丝附着的半桥粒 到基底外侧质膜，锚定细丝 似乎桥接半桥粒和致密层 基底膜和锚定原纤维网络 通过物理方式将致密层附着到下方的真皮 真皮纤维成分的截留。 失败这个 附着复合物会导致多种形式的起泡 疾病统称为大疱性表皮松解症（EB）。 虽然 真皮与表皮分离的现象已有充分记录， 导致这些分离的分子事件不是 明确知道。 在过去三年的资助中， 这笔赠款支持了一些研究，这些研究确定了大型和 VII 型胶原 C 末端球状结构域 (NC-1) 并表示该领域参与了新的模型 锚定原纤维网络。 该模型基于结构 VII 型胶原蛋白的表征和超微结构 NC-1 的免疫定位。 该模型预测了相互作用 NC-1 本身、IV 型胶原蛋白和其他内在成分 基底膜成分与致密层和 在结构中我们称之为“锚定板”。 本申请建议继续进行这些研究：(1) 研究 NC-1 与其自身以及与 IV 型胶原蛋白和其他基底膜的结构域 使用固相相互作用分析和生物物理分析蛋白质 技术； (2) 尝试分离额外的 NC-1 结合 通过亲和层析分析蛋白质并表征其 结构； (3) 开始结构鉴定 通过首先产生单克隆抗体来制备半桥粒的成分 富集膜组分的抗体及其用途 这些抗体用于免疫分离和结构 他们的特征； (4) 评估培养者的能力 EB 角质形成细胞使用 cDNA 合成 VII 型胶原蛋白 杂交和免疫印迹技术，并评估 隐性遗传个体的锚定原纤维网络有缺陷 相对于上述模型的营养不良 EB，使用 电子显微镜免疫组织化学。