INTRACELLULAR MECHANICS OF INTESTINAL MUCUS SECRETION

该项目的长期目标是了解 调节粘蛋白生物合成的机制 肠杯状细胞和确定粘蛋白如何改变， 包装、运输和隐藏。 这些研究将有助于 表征粘蛋白在疾病状态下如何改变， 囊性纤维化，CF，并最终有助于更好的临床 管理混乱。 在水流的范围内 根据建议，将采取几种方法。 第一，研究 杯状细胞分泌的膜动力学将扩大， 包括在手术过程中和手术过程中杯状细胞的形态测定分析， 在粘液快速释放之后。 膜流也将是 通过电子显微镜水平的示踪剂研究表征 使用电子密度标记。 膜改变后， 分泌物将以冷冻断裂为特征。 第二、 研究，以确定三维构成的 杯状细胞的高尔基体区域的细胞骨架将被 继续使用海卫一的薄切片系列重建 模型以及快速冻结，深蚀刻，旋转阴影复制品。 第三，参与刺激/分泌的细胞内步骤 偶联将在分泌粘蛋白的人中表征， 癌细胞系，T-84，使用免疫测定法， 多克隆或特异性单克隆抗体。 第四，含有粘蛋白颗粒的T-84细胞中的氯离子通道 并且分离的大鼠杯状细胞将以斑片- 夹紧技术。 最后，将使用形态测量模型 为了检测在细胞中合成的粘蛋白种类的改变， 对药理学操作的反应。 这些更改将 使用特异性单克隆抗体通过免疫细胞化学监测 抗鼠粘蛋白抗体。 这些研究将相互关联 用碳水化合物细胞化学和荧光染色- 标记的凝集素，并将作为使用cDNA研究的基础 probes. 了解杯状细胞如何响应药理学 代理商和不同的分泌状态将有助于我们的理解 粘液在肠道生物学中的作用。 它将 并提供必要的背景资料， 可用于涉及此的疾病的临床管理 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.