Seagle Brandon-Luke L, Gasyna Elzbieta M, Mieler William F, Norris James R
Department of Chemistry, University of Chicago, 5735 South Ellis Avenue, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2006 Nov 7;103(45):16644-8. doi: 10.1073/pnas.0605986103. Epub 2006 Oct 30.
Cultured retinal pigment epithelium (RPE) cells can phagocytize large foreign particles. Heterogeneous melanin aggregates from Sepia officinalis, a species of cuttlefish, were fed to cultured human RPE cells to produce cells laden with Sepia melanin. Blue light-induced apoptosis (BLIA) assays were performed by flow cytometry on parallel cultures consisting of RPE cells isolated from independent eyes and evenly divided into two cultures, one fed Sepia melanin and one containing only native melanin. After culturing and growth of the cells under blue light illumination for 7 days, the apoptosis percentage of all cultures indicated that Sepia feeding significantly reduced BLIA. To account for Sepia photoprotection, continuous-wave EPR and time-resolved EPR experiments were performed with parallel RPE cultures by using UV (355 nm) and green (532 nm) laser irradiation. Continuous-wave EPR spectra prove that the concentrations of intrinsic and extrinsic melanin free radicals in the Sepia-RPE culture are large compared with those concentrations in the RPE culture. Time-resolved EPR spectra indicate that both UV and green light produced extrinsic melanin radicals as radical pairs from the triplet manifold with a linear dependence on the number of photons per second. These experiments conclusively demonstrate that decreased RPE susceptibility to BLIA correlates with increased intracellular melanin free radical concentrations and that nonnative melanin can supplement native melanin photoprotection of RPE cells.
培养的视网膜色素上皮（RPE）细胞能够吞噬大型外来颗粒。将来自乌贼（一种墨鱼）的异质黑色素聚集体喂食给培养的人RPE细胞，以产生负载乌贼黑色素的细胞。通过流式细胞术对平行培养物进行蓝光诱导凋亡（BLIA）分析，这些平行培养物由从独立眼睛分离的RPE细胞组成，并均匀分为两种培养物，一种喂食乌贼黑色素，另一种仅含有天然黑色素。在蓝光照射下培养和生长7天后，所有培养物的凋亡百分比表明，喂食乌贼黑色素显著降低了BLIA。为了解释乌贼的光保护作用，通过使用紫外（355nm）和绿色（532nm）激光照射，对平行的RPE培养物进行了连续波EPR和时间分辨EPR实验。连续波EPR光谱证明，与RPE培养物中的浓度相比，乌贼-RPE培养物中内在和外在黑色素自由基的浓度较高。时间分辨EPR光谱表明，紫外光和绿光均产生外在黑色素自由基，作为来自三重态的自由基对，与每秒光子数呈线性相关。这些实验最终证明，RPE对BLIA的敏感性降低与细胞内黑色素自由基浓度增加相关，并且非天然黑色素可以补充RPE细胞的天然黑色素光保护作用。
