ENERGY METABOLISM OF CULTURED HUMAN RPE

培养人类 RPE 的能量代谢

• 批准号: 3263417
• 负责人: Michael V Miceli
• 金额: $ 9.23万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3263417
• 关键词: bioenergetics; cellular respiration; diabetic retinopathy; electron transport; glucose; glycolysis; human age group; human tissue; lactates; nuclear magnetic resonance spectroscopy; phosphorus compounds; radiotracer; retina degeneration; retinal pigment epithelium; retinaldehyde; retinitis pigmentosa; tissue /cell culture

The long term objective is to measure the energy demand of cultured human retinal pigment epithelium (RPE) and determine how this varies with different culture conditions or source of culture material. The specific aims are to determine: 1) the glycolytic flux and respiratory activity of normal cultured RPE; 2) how this changes with the density of cells in culture i.e. from pre-confluent, to confluent, to post confluent cultures; 3) the effect of donor age, and of passage number on these activities; 4) what differences, if any, exist in the energy pathways of RPE cells cultured from eyes with diabetic retinopathy, age related macular degeneration, and retinitis pigmentosa as an indication of changes in energy production and energy demand; and 5) the relationship between the major organophosphate metabolites observable in the phosphorus-31 (P-31) nuclear magnetic resonance (NMR) spectrum of RPE cells to the glycolytic and respiratory activity of the cell. In order to achieve these goals RPE will be cultured from human donor eyes and expanded in culture. Morphology will be monitored by phase contrast light microscopy. The cells will also be characterized by cell doubling times. Quantitative measurements of glucose utilization and lactate production in RPE will be made with Carbon-13 (C-13) labeled glucose using C-13 NMR spectroscopy. Cells will be maintained in the NMR magnet by casting cells in agarose threads and perfusing medium past the cells. The organophosphate metabolites and stability of the preparation will be monitored using P-31 NMR. Oxygen consumption of the cells will be measured using an oxygen electrode in separate experiments. This data will increase our knowledge of RPE energy metabolism and determine if RPE from eyes with known retinal degenerations have altered energy production or energy demand. It is also hoped that the relationship between the major phosphorus metabolites and energy metabolism will provide specific metabolic markers which will be useful in the future development of techniques to examine RPE metabolism in vivo.

长期目标是衡量培养的人类的能源需求 视网膜色素上皮（RPE），并确定它如何随 不同的培养条件或培养物质的来源。 具体 目的是确定：1​​） 正常培养的RPE； 2）这如何随细胞密度而变化 培养，即从汇聚，到融合到融合文化； 3）捐赠者年龄和通过数量对这些活动的影响； 4） RPE细胞的能量途径中存在哪些差异（如果有的话） 与糖尿病性视网膜病的眼睛培养，与年龄相关的黄斑 变性和色素性视网膜炎作为变化的指示 能源生产和能源需求； 5） 磷-31中可观察到的主要有机磷酸代谢物（P-31） RPE细胞的核磁共振（NMR）光谱 和细胞的呼吸活性。 为了实现这些目标RPE 将从人类的捐助者眼中培养，并在文化中扩展。 形态学 将通过相对比光学显微镜监测。 细胞也将 以细胞加倍时间为特征。 定量测量 RPE中的葡萄糖利用率和乳酸产生将与 碳13（C-13）使用C-13 NMR光谱标记葡萄糖。 细胞会 通过在琼脂糖螺纹中铸造细胞和 使中等范围经过细胞。 有机磷酸盐代谢物和 制剂的稳定性将使用P-31 NMR监测。氧 细胞的消耗将使用氧电极在 单独的实验。 这些数据将增加我们对RPE能源的了解 代谢，并确定已知视网膜变性的眼睛的RPE是否 能源生产或能源需求改变了。 也希望 主要磷代谢物与能量代谢之间的关系 将提供特定的代谢标记，将来有用 开发在体内检查RPE代谢的技术。