INTRACELLULAR MECHANICS OF INTESTINAL MUCUS SECRETION

肠粘液分泌的细胞内机制

• 批准号: 3232119
• 负责人: ROBERT D SPECIAN
• 金额: $ 8.08万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3232119
• 关键词: Golgi apparatus; apical membrane; autoradiography; complementary DNA; cystic fibrosis; enzyme linked immunosorbent assay; gastrointestinal epithelium; histochemistry /cytochemistry; immunochemistry; immunocytochemistry; intestinal mucosa; intracellular membranes; laboratory mouse; laboratory rabbit; laboratory rat; microtubules; monoclonal antibody; mucins; organ culture; orphan disease /drug; secretion

The long-term goal of this project is to understand the precise regulatory mechanisms governing the biosynthesis of mucins in intestinal goblet cells and to define how the mucins are altered, packaged, transported and secreted. These studies will help characterize how mucins are altered in disease states such as cystic fibrosis, CF and eventually contribute to the better clinical management of the disorder. Within the confines of the current proposal, several approaches will be taken. First, studies on the membrane dynamics of goblet cell secretion will be expanded, including a morphometric analysis of goblet cells during and following the rapid release of mucus. Membrane flow will also be characterized by tracer studies at the electron microscopic level using electron dense markers. Membrane alterations after secretion will be characterized by freeze fracture. Second, studies to define the three dimensional makeup of the cytoskeleton in the Golgi region of the goblet cell will be continued using thin section serial reconstructions of Triton models as well as rapid-freeze, deep-etch, rotary-shadow replicas. Third, the intracellular steps involved in stimulus/secretion coupling will be characterized in a mucin-secreting human carcinoma cell line, T-84, using an immunoassay employing a polyclonal or specific monoclonal antibodies against human mucin. Fourth, chloride channels in T-84 cells containing mucin granules and isolated rat goblet cells will be characterized by patch- clamping techniques. Finally, a morphometric model will be used to detect alterations in the mucin species being synthesis in response to pharmacologic manipulation. These changes will be monitored by immunocytochemistry using specific monoclonal antibodies against rat mucin. These studies will be correlated with carbohydrate cytochemistry and staining with fluorescently- labeled lectins and will serve as a basis for studies using cDNA probes. Understanding how goblet cells respond to pharmacologic agents and to varied secretory states will aid in our understanding of the role of mucus in the biology of the intestinal tract. It will also provide necessary background information that may prove useful in the clinical management of disorders which involve this glycoprotein.

该项目的长期目标是了解精确的 管理粘蛋白生物合成的调节机制 肠道杯细胞并定义粘蛋白的改变， 包装，运输和分泌。 这些研究将有助于 表征粘蛋白如何改变疾病状态（例如 囊性纤维化，CF，最终有助于更好的临床 疾病的管理。 在电流的范围内 提案，将采取几种方法。 首先，研究 杯状细胞分泌的膜动力学将扩展， 包括对杯状细胞的形态分析和 粘液迅速释放之后。 膜流也将是 以电子显微镜水平的示踪剂研究为特征 使用电子致密标记。 之后的膜改变 分泌的特征是冻结。 第二， 研究定义三维构成的研究 杯状细胞的高尔基区域的细胞骨架将是 继续使用Triton的薄部分串行重建 模型以及快速冻结，深蚀刻，旋转阴影复制品。 第三，刺激/分泌所涉及的细胞内步骤 耦合将以粘蛋白分泌的人来表征 癌细胞系T-84，使用采用A的免疫测定 对人粘蛋白的多克隆或特异性单克隆抗体。 第四，含有粘蛋白颗粒的T-84细胞中的氯化物通道 和分离的大鼠杯状细胞将以斑块为特征 夹紧技术。 最后，将使用形态计量学模型 检测粘蛋白物种的改变是合成的 对药理操作的反应。 这些更改将是 使用特定的单克隆人通过免疫细胞化学监测 抗大鼠粘蛋白的抗体。 这些研究将相关 与碳水化合物细胞化学和荧光染色 标记为凝集素，并将作为使用cDNA的研究的基础 探针。 了解杯状细胞对药物的反应 代理人和多样化的分泌国家将有助于我们的理解 粘液在肠道生物学中的作用。 会 还提供可能证明的必要背景信息 在涉及此的疾病的临床管理中有用 糖蛋白。