PHYSIOLOGY OF EPITHELIAL MONOLAYERS GROWN IN VITRO

当MDCK细胞(来源于正常肾脏的上皮样细胞系) DOG)是通过对先前的单分子层进行胰酶消化并在高温下镀制而成的 在可渗透和半透光的支撑物上的密度，它们形成了 不间断的单层，具有许多结构和功能 上皮细胞转运的特点。这些产品的两个主要特点 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 障碍物；2)顶端和基底侧面是结构性的和 有趣的不同，表明在自然运输中 上皮细胞，细胞极化。这些特征如图15所示 电镀后数小时。我们的总体目标是探索这些 所安装的特性以及此特定功能的具体目的 项目是：1)研究建立新的闭合交叉口 电镀细胞，并与融合单层中的建立进行比较 在没有钙的情况下保存了20小时，我们知道的情况是 从未封闭过的接头；2)用免疫荧光进行研究 抗ATPase抗体是否极化到根尖/基底外侧 元素取决于遮挡连接的形成；3)至 用微电极和扫描电子显微镜表征离子迁移机制 测量示踪剂通量。这将在稳定状态的单层中完成 (培养超过20h)，在刚刚培养的那些中，胰酶 可能已经分解了相关的蛋白质，给了我们一个追踪的机会 机制是如何树脂和重新安装的；4)我们有一个突变体 耐高剂量哇巴因的MDCK细胞。我们想要学习 离子转运机制、~3H哇巴因结合等，以了解它们为什么 抵抗。5)当普通的、野生的MDCK细胞混合培养时 与突变体形成单层，它就会受到哇巴因的保护。我们计划 阐明这种保护的机制。

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.