Human Retinal Pigment Epithelial Cell Cultures: Physiology & Fluid Transport

眼病如老年性黄斑变性或糖尿病影响视网膜色素上皮（RPE）功能，导致视网膜变性、视力丧失和失明。为了研究RPE的功能、生理和病理，许多实验室试图培养RPE作为天然组织更容易获得的替代品。由于RPE及其在视网膜和脉络膜中的邻近细胞的功能和形态复杂性，这一目标已经取得了不同程度的成功。我们已经开发了培养人类胎儿RPE细胞融合单层的技术，这些细胞表现出与天然人类胎儿RPE相似的形态、生理学和蛋白质表达模式。这项工作的目标之一是确定一组商业上可用的成分，以创建稳定和可重复的RPE细胞培养。我们能够产生具有经典上皮形态的融合色素RPE细胞培养物，经皮电位为1 - 3mV，经皮电阻大于400欧姆*cm2。在本实验中，我们使用电子显微镜和免疫组织化学进一步表征这些培养物，以鉴定细胞结构，并定位根尖和基底外侧膜和细胞间连接复合物蛋白。采用elisa法确定所选细胞因子的分泌极性。细胞内微电极用于表征受体介导的第二信使通路及其在根尖和基底膜上的下游电生理特性。电容探针技术用于测量净上皮液体传输。该模型已被用于帮助确定一些线粒体途径，介导氧化应激和硫辛酸的保护作用。定义了RPE的基因特征

Eye diseases such as age-related macular degeneration or diabetes affect retinal pigment epithelium (RPE) function and lead to retinal degeneration, vision loss and blindness. To study RPE function, physiology, and pathology, many laboratories have attempted to culture RPE as a more accessible alternative to native tissue. This goal has been accomplished with varying degrees of success due to the functional and morphological complexity of the RPE and its neighboring cells in the retina and the choroid. We have developed techniques for culturing confluent monolayers of human fetal RPE cells that exhibit morphology, physiology, and patterns of protein expression similar to native human fetal RPE. One of the goals of this work was to identify a set of commercially available ingredients to create stable and reproducible RPE cell cultures. We been able to produce confluent pigmented RPE cell cultures with classic epithelial morphology, transepithelial potential of 1 - 3mV, and transepithelial resistance greater than 400 Ohms*cm2. In the present experiments we further characterized these cultures using electron-microscopy and immunohistochemistry to identify cell structures, and localize apical and basolateral membrane and intercellular junctional complex proteins. ELISAs were used to confirm the polarity of secretion of selected cytokines. Intracellular microelectrodes were used to characterize receptor-mediated second messenger pathways and their downstream electrophysiological properties at the apical and basolateral membranes. The capacitance probe technique was used to measure net transepithelial fluid transport. This model has been used to help identify some of the mitochondrial pathways that mediate oxidative stress and the protective effects of lipoic acid. Gene signature of RPE was defined