The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.
The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.
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发表时间：
2000-06
影响因子：
4.4
通讯作者：
J. Sparrow;Koji Nakanishi;C. Parish
中科院分区：
文献类型：
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作者：
J. Sparrow;Koji Nakanishi;C. Parish
目的 确定脂褐素荧光团 A2E 是否参与蓝光诱导的视网膜色素上皮 (RPE) 细胞损伤。方法 人 RPE 细胞 (ARPE-19) 在培养基中积累浓度为 10、50 和 100 µM 的 A2E，通过定量高效液相色谱 (HPLC) 测定，内化 A2E 的水平范围为低于 5 至 64 ng/10(5) 细胞。随后将汇合培养物的限制区（直径为 0.5 毫米的斑点）暴露于 480 +/- 20 纳米（蓝色）或 545 +/- 1 纳米（绿色）光下 15 至 60 秒。通过膜受损细胞核的荧光染色、凋亡细胞的 TdT-dUTP 末端切口标记 (TUNEL) 以及磷脂酰丝氨酸暴露的膜联蛋白 V 标记来量化暴露后不同时期的光毒性。结果非存活细胞位于含有 A2E 的 RPE 细胞的蓝光暴露区域，而位于照明区域之外的细胞仍然存活。如膜损伤细胞核的荧光标记和 TUNEL 阳性细胞的存在所示，非存活细胞的数量随着暴露时间的延长而增加，并且是光照前用于加载细胞的 A2E 浓度的函数。蓝光诱导的 TUNEL 阳性细胞数量也在膜受损细胞标记增加之前增加，这一发现与膜联蛋白 V 标记一起表明细胞死亡的凋亡形式。相反，不含 A2E 的蓝光暴露 RPE 细胞仍然存活。此外，用绿光照射导致出现的非存活细胞显着减少。结论 这些研究表明 A2E 是蓝光诱导 RPE 细胞凋亡的引发剂。
PURPOSE To determine whether the lipofuscin fluorophore A2E participates in blue light-induced damage to retinal pigmented epithelial (RPE) cells. METHODS Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 microM concentrations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/10(5) cells, as assayed by quantitative high-performance liquid chromatography (HPLC). Restricted zones (0.5-mm diameter spots) of confluent cultures were subsequently exposed to 480 +/- 20-nm (blue) or 545 +/- 1-nm (green) light for 15 to 60 seconds. Phototoxicity was quantified at various periods after exposure by fluorescence staining of the nuclei of membrane-compromised cells, by TdT-dUTP terminal nick-end labeling (TUNEL) of apoptotic cells and by Annexin V labeling for phosphatidylserine exposure. RESULTS Nonviable cells were located in blue light- exposed zones of A2E-containing RPE cells, whereas cells situated outside the illuminated areas remained viable. As shown by fluorescence labeling of the nuclei of membrane-damaged cells and by the presence of TUNEL-positive cells, the numbers of nonviable cells increased with exposure duration and as a function of the concentration of A2E used to load the cells before illumination. The numbers of blue light-induced TUNEL-positive cells also increased in advance of the increase in labeling of membrane-compromised cells, a finding that, together with Annexin V labeling, indicates an apoptotic form of cell death. Conversely, blue light- exposed RPE cells that did not contain A2E remained viable. In addition, illumination with green light resulted in the appearance of substantially fewer nonviable cells. CONCLUSIONS These studies implicate A2E as an initiator of blue light-induced apoptosis of RPE cells.