INTERMEDIARY METABOLISM OF HUMAN RPE CELLS

长期目标是测量培养的人类的能量需求。 视网膜色素上皮(RPE)，并确定这种变化如何与 不同的培养条件或培养材料的来源。具体的 目的是测定：1)糖酵解通量和呼吸活性 正常培养的RPE；2)随着细胞密度的变化 文化，即从融合前的文化，到融合的文化，再到融合的文化； 3)供者年龄和代次对这些活动的影响；4) RPE细胞的能量通路是否存在差异？ 从糖尿病视网膜病变、老年性黄斑病变的眼睛培养 变性和视网膜色素变性作为变化的迹象 能源生产与能源需求；5)能源生产与能源需求 磷-31(P-31)中可观察到的主要有机磷代谢产物 RPE细胞对糖酵解的核磁共振波谱 以及细胞的呼吸活动。为了实现这些目标，RPE 将从人类捐赠者的眼睛中培养并在培养中扩增。形态学 将通过相差光学显微镜进行监测。这些细胞也会 以细胞倍增时间为特征。定量测量 RPE中葡萄糖的利用和乳酸的生产将由 碳-13(C-13)标记葡萄糖的碳-13核磁共振波谱。单元格 通过在琼脂糖线中铸造细胞和在核磁共振磁体中保持 向细胞外灌流培养液。有机磷代谢物和 该制剂的稳定性将使用P-31核磁共振进行监测。氧 电池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能量的了解 已知视网膜退行性变患者的RPE的代谢和检测 改变了能源生产或能源需求。同时也希望 主要磷代谢产物与能量代谢的关系 将提供特定的代谢标记，这在未来将是有用的 体内RPE代谢检测技术的发展。

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.