ENERGY METABOLISM OF CULTURED HUMAN RPE

长期目标是衡量有文化的人类的能源需求 视网膜色素上皮 (RPE) 并确定其如何随 不同的培养条件或培养材料来源。 具体的 目的是确定： 1) 糖酵解通量和呼吸活动 正常培养的 RPE； 2）这如何随着细胞密度的变化而变化 培养，即从汇合前、汇合、汇合后培养； 3) 供体年龄和传代次数对这些活动的影响； 4） RPE 细胞的能量途径存在哪些差异（如果有） 从患有糖尿病视网膜病变、年龄相关性黄斑的眼睛中培养 变性和色素性视网膜炎作为变化的指标 能源生产和能源需求； 5) 之间的关系 在磷-31 (P-31) 中可观察到的主要有机磷代谢物 RPE 细胞糖酵解的核磁共振 (NMR) 谱 和细胞的呼吸活动。 为了实现这些目标 RPE 将从人类捐赠者的眼睛中培养并在文化中扩展。 形态学 将通过相差光学显微镜进行监测。 细胞也会 通过细胞倍增时间来表征。 定量测量 RPE 中的葡萄糖利用和乳酸生产将通过 使用 C-13 NMR 光谱法碳 13 (C-13) 标记葡萄糖。 细胞会 通过将细胞浇铸在琼脂糖线中来维持在 NMR 磁体中，并且 灌注培养基经过细胞。 有机磷代谢物和 使用 P-31 NMR 监测制剂的稳定性。氧气 电池的消耗将使用氧电极测量 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 RPE 是否来自患有已知视网膜变性的眼睛 改变了能源生产或能源需求。 还希望 主要磷代谢物与能量代谢的关系 将提供将来有用的特定代谢标记 开发检测体内 RPE 代谢的技术。

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.