INTRACELLULAR MECHANICS OF INTESTINAL MUCUS SECRETION

这个项目的长期目标是了解准确的 粘蛋白生物合成的调控机制 肠道杯状细胞和确定粘蛋白是如何改变的， 包装、运输和保密。这些研究将会有所帮助 描述在疾病状态下粘蛋白是如何改变的，例如 囊性纤维化、CF并最终有助于改善临床 无序的管理。在海流的范围内 关于这一提议，将采取几种办法。第一，关于中国经济社会发展的研究 杯状细胞分泌物的膜动力学将扩大， 包括杯状细胞的形态计量分析 在粘液迅速释放之后。膜流量也将被 以电子显微镜水平上的示踪研究为特征的 使用电子密度标记。术后的细胞膜改变 分泌物将以冷冻断裂为特征。第二, 研究定义女性的三维构成 杯状细胞高尔基体区的细胞骨架 继续使用Triton的薄层连续重建 模型以及快速冷冻、深度蚀刻、旋转阴影复制品。 第三，刺激/分泌所涉及的细胞内步骤 在分泌粘蛋白的人类中，偶联将成为特征 肿瘤细胞株T-84的免疫分析 抗人粘蛋白的多克隆或特异性单抗。 含粘蛋白颗粒的T-84细胞的氯离子通道 而分离的大鼠杯状细胞将通过贴片- 夹紧技术。最后，将使用形态测量模型 检测正在合成的粘蛋白种类的变化 对药物操纵的反应。这些变化将是 用特定的单抗进行免疫细胞化学监测 抗大鼠粘蛋白抗体。这些研究将是相互关联的 用碳水化合物细胞化学和荧光染色- 标记的凝集素，将作为使用cdna进行研究的基础。 探测器。了解杯状细胞对药物的反应 代理人和不同的分泌状态将有助于我们理解 粘液在肠道生物学中的作用。会的 还提供必要的背景信息，以证明 对涉及这种疾病的疾病的临床治疗很有用 糖蛋白。

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.