CELL SURFACE PROTEINS OF CULTURED ORAL KERATINOCYTES

口腔上皮细胞处于重要的界面位置。 组织和环境，并参与各种相互作用 唾液、病原体、邻近细胞和细胞外基质 蛋白质。这项建议涉及到分配和类型的 口腔角质形成细胞表面存在受体(如层粘连蛋白和 纤维连接蛋白受体)及其生物发生和一种可能的 角质形成细胞分泌。细胞将在双室中生长为融合。 培养系统，允许分别访问根尖和根尖上的培养液 基面。结构域选择性生物素化偶联 用固定化凝集素沉淀将用于分析根尖 和基础糖蛋白的分布。上皮细胞的生物发生 糖蛋白的研究将结合代谢放射性标记和 结构域选择性生物素化用于监测药物的传递 新合成的糖蛋白进入细胞表面。单层将是 脉冲(35)S-蛋氨酸并以不同的间隔追赶以允许 糖蛋白修饰和到达顶端和基底区。 用S-NHS-生物素进行结构域选择性标记后，细胞裂解， TX-114提取，放射性标记/生物素标记的蛋白质 用固定化链霉亲和素回收。或者，心尖部和 基底表面将暴露于生物素-肼以直接标记 糖蛋白及其加工半薄冰冻切片的单层 和免疫荧光。生物素标记的角质形成细胞膜蛋白 释放到心尖室和基底室的蛋白质 通过一维和二维电泳分析，等聚焦， 荧光照相和蛋白质印迹。所提出的体外模型的应用 口腔角质形成细胞生物学研究系统可能提供有价值的 信息：微生物和病毒的受体， 细胞外基质蛋白、细胞因子释放、跨上皮 分泌免疫球蛋白A的递送，以及细胞的黏附和迁移。这个 这项研究的长期目标是了解 口腔角质形成细胞表面与细胞间通讯有关 口腔黏膜病的病因学。

Oral epithelial cells are strategically located at the interface of tissues and environment and are involved in a variety of interactions with saliva, pathogens, adjacent cells, and extracellular matrix proteins. This proposal deals with the distribution and the kind of receptors present on the oral keratinocyte cell surface (e.g. laminin and fibronectin receptors) as well as their biogenesis and a putative keratinocyte secretion. Cells will be grown to confluence in a bicameral culture system to allow separate access to the media on the apical and basal surfaces. Domain-selective biotinylation coupled with precipitation with immobilized lectins will be used to analyze the apical and basal glycoprotein distribution. The biogenesis of epithelial glycoproteins will be studied by combining metabolic radiolabeling and domain-selective biotinylation to monitor the delivery of newly-synthesized glycoproteins to the cell surface. Monolayers will be pulsed with (35)S-methionine and chased at different intervals to permit glycoprotein modifications and arrival at the apical and basal domains. After domain-selective labeling with S-NHS-biotin, cells will be lysed, extracted with TX-114, and the radiolabeled/ biotinylated proteins recovered with immobilized streptavidin. Alternatively, the apical and basal surfaces will be exposed to biotin-hydrazide to directly label glycoproteins and the monolayers processed for semithin frozen sections and immunofluorescence. The biotinylated keratinocyte membrane proteins and the proteins released to the apical and basal compartments will be analyzed by one- and two-dimensional electrophoresis, isofocusing, fluorography, and western-blotting. Use of the proposed in vitro model system for studying oral keratinocyte biology may provide valuable information on: receptors for microorganisms and viruses, receptors for extracellular matrix proteins, cytokines release, transepithelial delivery of secretory IgA, and cell adhesion and migration. The long-term goals of this research are to understand how changes in the oral keratinocyte surface relate to cell-to-cell communication and the etiology of oral mucosa diseases.