PHYSIOLOGY OF EPITHELIAL MONOLAYERS GROWN IN VITRO

体外生长的单层上皮的生理学

• 批准号: 3227906
• 负责人: MARCELINO CEREIJIDO
• 金额: $ 1.87万
• 依托单位: NATIONAL POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-02-01 至 1987-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3227906
• 关键词: adenosinetriphosphatase; basolateral membrane; cell bank /registry; epithelium; ion transport; kidney cell; membrane permeability; membrane structure; ouabain; tissue /cell culture; trypsin

When MDCK cells (an epithelioid line derived from the kidney of a normal dog) is obtained by trypsination of previous monolayers and plated at high densities on a permeable and transluscent support, they develop an uninterrupted monolayer which has many of the structural and functional characteristics of transporting epithelia. The main two features of these cells are: 1) they contact each other and form occluding junctions which confer to the monolayers the ability to act as effective permeability barriers; and 2) the apical and the basolateral aspects are structural and funcionally different, indicating that, as in natural transporting epithelia, cells are polarized. These characteristics are exhibited 15 hours after plating. Our overall aim is to explore how these characteristics are installed and, the specific aim of this particular project is: 1) To study the establishment of occluding junctions in newly plated cells, and compare it with the establishment in confluent monolayers that have been kept without Ca for 20 h, situation where we know that junctions have never been sealed; 2) To study with immunofluorescent antibodies against ATPase whether polarization into apical/basolateral elements depends on the formation of occluding junctions; 3) To characterize the ion translocating mechanisms by using microelectrodes and measuring tracer fluxes. This will be done in steady state monolayers (plated for more than 20 h.), and in those just plated, in which trypsin might have cleaved the proteins involved, and give us a chance to follow how are the mechanisms resinthesized and reinstalled; 4) We have a mutant of MDCK cell which is resistant to high dosis of ouabain. We want to study ion translocating mechanisms, 3H-ouabain binding, etc. to learn why they resist. 5) When the common, wild MDCK cell, is co-cultured in mixed monolayer with the mutant, it is protected from ouabain. We plan to elucidate the mechanisms of this protection.

当MDCK细胞（一种来源于正常人肾脏的上皮样细胞系） 狗）通过胰蛋白酶消化先前的单层获得，并以高浓度铺板。 在可渗透的和无色的载体上，它们形成一种 不间断的单层，具有许多结构和功能 运输上皮细胞的特征。 这两个主要特点 细胞是：1）它们彼此接触并形成闭塞连接， 赋予单层作为有效渗透性的能力 屏障;和2）顶端和基底外侧方面是结构性的， 功能不同，表明在自然运输中， 上皮细胞是极化的。 这些特点表现在15 在镀覆后数小时。 我们的总体目标是探索这些 安装的特点，并在此特定的目标， 本课题的主要内容是：1）研究在新生儿中建立闭合连接， 平板细胞，并将其与融合单层中的建立进行比较 在没有钙的情况下保持20小时，我们知道， 2）免疫荧光法研究 抗ATP酶抗体是否极化至顶侧/基底侧 元素取决于闭塞连接的形成; 3） 利用微电极表征离子迁移机制， 测量示踪剂通量。 这将在稳态单层中进行 （平板培养20小时以上），而在那些刚刚铺板的， 可能已经切割了相关的蛋白质，给了我们一个机会， 这些机制是如何重新合成和重新安装的; 4）我们有一个突变体 MDCK细胞对高剂量哇巴因具有耐药性。 我们要研究 离子转运机制，3 H-哇巴因结合等，以了解为什么它们 抗拒. 5)当普通的野生MDCK细胞在混合培养基中共培养时， 单层与突变体，它是保护从哇巴因。 我们计划 阐明这种保护的机制。