Gao Fangyuan, Tom Emily, Lieffrig Stephanie A, Finnemann Silvia C, Skowronska-Krawczyk Dorota
Department of Ophthalmology, Center for Translational Vision Research, School of Medicine, UC Irvine, Irvine, CA, United States.
Department of Physiology and Biophysics, Department of Ophthalmology, Center for Translational Vision Research, School of Medicine, UC Irvine, Irvine, CA, United States.
Front Mol Neurosci. 2023 Oct 20;16:1279457. doi: 10.3389/fnmol.2023.1279457. eCollection 2023.
The vertebrate retinal pigment epithelium (RPE) lies adjacent to the photoreceptors and is responsible for the engulfment and degradation of shed photoreceptor outer segment fragments (POS) through receptor-mediated phagocytosis. Phagocytosis of POS is critical for maintaining photoreceptor function and is a key indicator of RPE functionality. Popular established methods to assess RPE phagocytosis rely mainly on quantifying POS proteins, especially their most abundant protein rhodopsin, or on fluorescent dye conjugation of bulk, unspecified POS components. While these approaches are practical and quantitative, they fail to assess the fate of POS lipids, which make up about 50% of POS by dry weight and whose processing is essential for life-long functionality of RPE and retina.
We have developed a novel very-long-chain polyunsaturated fatty acids (VLC-PUFA)-based approach for evaluating RPE phagocytic activity by primary bovine and rat RPE and the human ARPE-19 cell line and validated its results using traditional methods.
This new approach can be used to detect the dynamic process of phagocytosis at varying POS concentrations and incubation times and offers a robust, unbiased, and reproducible assay that will have utility in studies of POS lipid processing.
脊椎动物视网膜色素上皮（RPE）与光感受器相邻，负责通过受体介导的吞噬作用吞噬和降解脱落的光感受器外段片段（POS）。POS的吞噬作用对于维持光感受器功能至关重要，并且是RPE功能的关键指标。常用的评估RPE吞噬作用的方法主要依赖于对POS蛋白进行定量，尤其是其最丰富的蛋白质视紫红质，或者对大量未指定的POS成分进行荧光染料偶联。虽然这些方法实用且定量，但它们无法评估POS脂质的命运，POS脂质占POS干重的约50%，其处理对于RPE和视网膜的终身功能至关重要。
我们开发了一种基于新型超长链多不饱和脂肪酸（VLC-PUFA）的方法，用于评估原代牛和大鼠RPE以及人ARPE-19细胞系的RPE吞噬活性，并使用传统方法验证了其结果。
这种新方法可用于检测不同POS浓度和孵育时间下吞噬作用的动态过程，并提供了一种强大、无偏差且可重复的检测方法，将在POS脂质处理研究中发挥作用。
