CHARACTERIZATION OF SUB-EPITHELIAL ANTIGENS

上皮下抗原的表征

• 批准号: 3157347
• 负责人: ROBERT E BURGESON
• 金额: $ 13.72万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 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/3157347
• 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.

人的皮肤角质形成细胞附着在下面的真皮上 矩阵通过复杂的附着机制，许多元素 这是外部针叶植物所独有的。其中包括 介导角蛋白细丝附着的半桥粒 到基侧质膜，锚定细丝， 似乎将半桥粒连接到大脑的致密层 基底膜和锚定纤维网络， 通过物理方法将致密层附着在真皮下 夹住真皮纤维成分。这是失败的 附着复合体导致几种形式的水泡 统称为大疱性表皮松解症(EB)。而当 真皮-表皮分离的现象被很好地记录下来， 导致这些分离的分子事件并不是 毫不含糊地知道。在过去三年的资助中， 这笔赠款支持了一些研究，这些研究确定了一个大型和 III型胶原复合体C末端球状结构域(NC-1) 并指出这个领域参与了一种新的 锚定原纤网。该模型基于结构 III型胶原蛋白的特性及超微结构 NC-1的免疫定位。该模型预测了 NC-1与自身、IV型胶原和其他固有的 基底膜成分包括致密层和 在结构内，我们将其称为“锚定斑块”。 本申请建议继续进行这些研究：(1) 研究NC-1与其自身以及与其之间可能的相互作用 IV型胶原和其他基底膜的结构域 蛋白质固相相互作用分析和生物物理研究 技术；(2)尝试分离额外的NC-1结合 蛋白质的亲和层析，并对它们的特性进行分析 结构；(3)开始识别结构 首次制备单抗制备半桥粒组分的研究 对它们富含的膜部分的抗体和使用 这些抗体用于免疫分离和结构 它们的特性；以及(4)评估培养的能力 EB角质形成细胞利用c DNA合成VII型胶原 杂交和免疫印迹技术，并评估 隐性遗传病个体的锚定纤维网络缺陷 营养不良EB相对于上述模型，使用 电子显微镜免疫组织化学。