CHARACTERIZATION OF SUB-EPITHELIAL ANTIGENS

上皮下抗原的表征

• 批准号: 3157346
• 负责人: ROBERT E BURGESON
• 金额: $ 13.31万
• 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 1993-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3157346
• 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.

人皮肤角质形成细胞附着于下层真皮 矩阵由一个复杂的连接机制，许多元素的 这是外部上皮细胞所特有的 其中包括 介导角蛋白丝附着的半桥粒 到基底外侧质膜，锚定丝， 似乎桥的半桥粒的致密板的 基底膜和锚定纤维网络， 通过物理方法将致密层附着于下jacket真皮 真皮纤维成分的截留。 失败的原因 附着复合物导致几种形式的起泡 这些疾病共同称为大疱性表皮病（EB）。 而 真皮-表皮分离的现象是有据可查的， 导致这些分离的分子事件 毫不含糊地知道。 在过去三年的资助中， 这项赠款支持了一些研究，这些研究确定了一个大的， VII型胶原复合物C末端球状结构域（NC-1） 并指出这一领域参与了一个新的模式， 锚定原纤维网络。 该模型基于结构 VII型胶原和超微结构的表征 NC-1的免疫定位。 该模型预测的相互作用 NC-1本身，IV型胶原蛋白和其他内在 基底膜成分既有致密层， 在我们称之为“锚定斑块”的结构内。 本申请建议继续进行这些研究，以：（1） 研究NC-1与自身以及与 IV型胶原和其他基底膜的结构域 使用固相相互作用测定和生物物理 技术;（2）尝试分离额外的NC-1结合 通过亲和色谱法分析蛋白质，并表征其 （3）开始识别结构 通过首次生产单克隆抗体来研究半桥粒的成分 针对富集了它们的膜级分的抗体以及它们的用途 这些抗体用于免疫隔离和结构 （4）培养人的能力; EB角质形成细胞利用cDNA合成VII型胶原 杂交和免疫印迹技术，并评估 有缺陷的锚定纤维网络的个人与隐性 相对于上述模型，使用 电镜免疫组化