GENESIS OF CELL MEMBRANE POLARITY IN CORNEAL EPITHELIUM

角膜上皮细胞膜极性的起源

• 批准号: 3264850
• 负责人: JOSE MARIO WOLOSIN
• 金额: $ 15.72万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3264850
• 关键词: adenosinetriphosphatase; apical membrane; basolateral membrane; beta adrenergic receptor; corneal epithelium; electron microscopy; electrophysiology; fluorescence; gene expression; genetic transcription; genetic translation; intercellular connection; laboratory rabbit; membrane activity; membrane proteins; membrane structure; molecular polarity; monoclonal antibody; radioassay; tissue /cell culture

The corneal epithelium is a polarized, multilayered tissue that fulfills, with high efficiency, a number of physiological and protective roles essential to corneal health while undergoing rapid cell replacement. To improve understanding of the physiological activities involved in the preservation of a normal epithelium, this 5-year study examines some of the fundamental events that underlie a) the replacement of cells at the surface of the cornea, and b) the maturation of membrane polarity of the cells during their migration through the epithelial strata. Essential to these studies is the recent development of a method to induce exfoliation of the epithelial cells on a layer-by-layer fashion. The method allows dissection of the epithelium into 5 distinguishable layers thereby facilitating the study of changes in a number of properties or functions as cells progress along the basal-to- surface stratification axis. The ultrastructure of tight junctions (t.j.) between mature surface cell will be determined by electron microscopy. Then, after induced exfoliation of these cells, the structural dynamics of t.j. assembly between the new cells at the surface will be characterized. The protein composition of t.j.'s and details of the intracellular mobilization of specific proteins t.j. assembly will be studied using a) a monoclonal antibody raised against an essential t.j. component, ovomorulin, and b) radiolabelling of nascent proteins during a protein synthesis (translation) dependent-phase of tight junction formation. The location of a translational step critical for t.j. assembly had already been established, studies to determine a transcriptional (mRNA synthesis) step are undertaken. Electrophysiological methods will be used to determine at which point during stratification C1- channels are inserted in the apical membrane. Scanning electron microscopy and a battery of fluorescent lectins will be used to study the maturation of the apical membrane microanatomy and surface expression of sugars. The concomitant changes in basolateral activities will be assessed by measuring the changes in Na++K+ ATPase in beta-adrenergic receptors. The effect of unfavorable environmental conditions on the surface cell replacement process will also be examined as well as the effect of certain biological variables on the maturation of membrane polarity in a cell culture system.

角膜上皮是极化的多层组织， 高效率地实现了许多生理和 对角膜健康至关重要的保护作用， 细胞置换 为了提高对生理学的理解， 参与正常上皮细胞保存的活动， 5-一年的研究审查了一些基本事件， 构成a）角膜表面细胞替换的基础， 和B）在培养过程中细胞膜极性的成熟 它们通过上皮层迁移。 对这些至关重要 研究是最近开发的一种方法， 上皮细胞以逐层方式脱落。 的 方法允许将上皮解剖成5个可区分的 层，从而促进在一些变化的研究， 当细胞沿着基底到基底沿着前进时， 表层成层轴 细胞紧密连接（t. j.）成熟表面之间 细胞将通过电子显微镜测定。 之后 诱导这些细胞的脱落，t. j. 新细胞在表面的组装将是 表征了 t. j.的蛋白质组成。的和细节 特异性蛋白质t. j.组装的细胞内动员将被 研究使用a）针对必需的 t. j.组分，卵球蛋白，和B）放射性标记新生 在蛋白质合成（翻译）依赖阶段的蛋白质 形成紧密连接。 平移步骤的位置 已经建立了对t. j.组装至关重要的研究， 确定转录（mRNA合成）步骤是 进行。 电生理学方法将用于 确定在分层期间C1通道在哪个点被 插入顶端膜中。 扫描电子显微镜 一组荧光凝集素将用于研究 根尖膜成熟的显微解剖和表面 糖的表达。 基底外侧的伴随变化 将通过测量Na++K+的变化来评估活性 β-肾上腺素能受体中的ATP酶。 不利影响 环境条件对表面细胞置换过程的影响 也将检查以及某些生物的影响， 细胞培养物中膜极性成熟的变量 系统