ENERGY METABOLISM OF CULTURED HUMAN RPE

长期目标是测量培养人类的能量需求 视网膜色素上皮细胞（RPE），并确定如何变化， 不同的培养条件或培养材料来源。 具体 目的是确定：1）糖酵解通量和呼吸活性 正常培养的RPE; 2）这如何随着细胞密度的变化， 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性的影响; 4） RPE细胞的能量通路存在什么差异，如果有的话， 从患有糖尿病性视网膜病变、年龄相关性黄斑 变性和色素性视网膜炎作为改变的指示， 能源生产和能源需求之间的关系; 5） 磷-31（P-31）中可观察到的主要有机磷代谢物 核磁共振（NMR）光谱的RPE细胞的糖酵解 和细胞的呼吸活动。 为了实现这些目标，RPE 将从人类供体眼睛中培养并在培养物中扩增。 形态 将通过相差光学显微镜进行监测。 这些细胞还将 以细胞倍增时间为特征。 的定量测量 葡萄糖的利用和乳酸盐的产生将与 使用C-13 NMR光谱法对碳-13（C-13）标记的葡萄糖进行检测。 细胞将 通过将细胞浇铸在琼脂糖线中而保持在NMR磁体中， 灌注培养基通过细胞。 有机磷代谢物和 将使用P-31 NMR监测制剂的稳定性。氧 将使用氧电极测量电池的消耗， 单独的实验 这些数据将增加我们对RPE能量的了解 代谢，并确定是否RPE从眼睛与已知的视网膜变性 改变了能源生产或能源需求。 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志物，这将在未来有用， 开发技术以检查体内RPE代谢。

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.