Human Retinal Pigment Epithelial Cell Cultures: Physiology & Fluid Transport

人视网膜色素上皮细胞培养：生理学

• 批准号: 7968352
• 负责人: Sheldon Miller
• 金额: $ 44.41万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968352
• 关键词: Affect; Age related macular degeneration; Apical; Blindness; Cell Culture Techniques; Cells; Cellular Structures; Choroid; Culture Techniques; Diabetes Mellitus; Electric Capacitance; Electron Microscopy; Epithelial; Exhibits; Eye diseases; Genes; Goals; Human; Immunohistochemistry; Laboratories; Lead; Liquid substance; Measures; Mediating; Methods; Microelectrodes; Mitochondria; Modeling; Molecular; Morphology; Oxidative Stress; Pathology; Pathway interactions; Pattern; Physiological; Physiology; Pigments; Property; Resistance; Retina; Retinal Degeneration; Second Messenger Systems; Structure of retinal pigment epithelium; Techniques; Thioctic Acid; Tissues; Work; basolateral membrane; cytokine; fetal; interest; monolayer; protective effect; protein complex; protein expression; receptor; research study; second messenger; success

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

老年性黄斑变性或糖尿病等眼病影响视网膜色素上皮(RPE)功能，导致视网膜变性、失明和失明。为了研究RPE的功能、生理学和病理学，许多实验室已经尝试将RPE培养为一种比天然组织更容易获得的替代品。由于RPE及其邻近的视网膜和脉络膜细胞在功能和形态上的复杂性，这一目标已经实现了不同程度的成功。我们已经开发了培养融合的人胎儿RPE细胞单层的技术，其形态、生理和蛋白质表达模式类似于天然的人胎儿RPE。这项工作的目标之一是确定一套商业上可用的成分，以创造稳定和可重复的RPE细胞培养。我们能够培养出融合的着色RPE细胞，具有经典的上皮形态，跨上皮电位为1-3 mV，跨上皮阻力大于400欧姆*cm2。在目前的实验中，我们使用电子显微镜和免疫组织化学进一步鉴定了这些培养物的特征，以确定细胞结构，并定位了顶端和基底侧膜以及细胞间连接复合体蛋白。用ELISA确定所选细胞因子分泌的极性。细胞内微电极被用来表征受体介导的第二信使通路及其在根尖膜和基底侧膜下游的电生理特性。采用电容探针法测定跨上皮液的净转运。这个模型已经被用来帮助识别一些介导氧化应激的线粒体途径和硫辛酸的保护作用。定义了RPE的基因特征