光诱导对人视网膜色素上皮细胞自噬和凋亡的影响

研究人视网膜色素上皮（RPE）细胞光损伤中自噬与凋亡之间的关系，并探讨自噬抑制剂3甲 基腺嘌呤（3MA）对光诱导下RPE细胞自噬和凋亡的影响。方法：实验研究。将体外培养的人RPE细 胞（ARPE-19）随机分为12 h对照组、12 h模型组、12 h 3MA组以及24 h对照组、24 h模型组、24 h 3MA组。对照组采用锡纸包裹避光培养；模型组接受光照刺激；3MA组中加入3 mmol/ml 3MA后接 受光照刺激，以自制三基色LED（发光二极管）冷光灯作为光源，在（16 500±500）lx光照强度下照 射细胞12 h和24 h。透射电子显微镜观察每组细胞超微结构，流式细胞仪测定细胞存活率，激光共 聚焦结合倒置荧光显微镜定性、定量分析自噬-溶酶体形态及荧光强度，Western blot法检测Beclin 1、 LC3和P62自噬相关蛋白表达量。数据采用单因素方差分析。结果：在光照12 h后，ARPE-19细胞自 噬水平可以抵抗光损伤，降低细胞凋亡率（F=931.53，P<0.001）；光照24 h后细胞自噬水平超过其正 常调节范围，细胞凋亡率升高，差异有统计学意义（F=1156.67，P<0.001）；使用3MA可抑制光诱导 的ARPE-19细胞过度自噬，降低细胞凋亡率（F=2.856，P=0.027），进而保护细胞以应对光损伤。结论： ARPE-19自噬水平随光照时间而变化；随着光照时间延长，细胞凋亡率升高。抑制细胞的过度自噬 可以保护ARPE-19细胞应对光损伤。

Effects of Light Induction on Autophagy and Apoptosis of Human Retinal Pigment Epithelial Cells

Objective: To study the effect of light damage on the relationship between autophagy and apoptosis in human retinal pigment epithelial (RPE) cells and to explore the effect of 3-methyladenine (3MA), an autophagy inhibitor, on light-induced autophagy and apoptosis in RPE cells. Methods: This experiment studied human RPE cells (ARPE-19) that were cultured in vitro and randomly divided into the following groups: 12 h control group, 12 h model group, 12 h 3MA group; 24 h control group, 24 h model group, and 24 h 3MA group. Tinfoil wrap was used in the control group to avoid light. The model group was stimulated by light, and the 3MA group received light stimulation after adding 3 mmol/mL 3MA. A custom made three-primary color LED (light-emitting diode) cold light lamp was used as the light source, and the cells were irradiated for 12 h and 24 h under 16 500±500 lux of light intensity. A transmission electron microscope was used to observe cell ultrastructure, flow cytometry was used to measure cell viability, a confocal laser combined with an inverted fluorescence microscope was used for the qualitative and quantitative analysis of autophagy-lysosome morphology and fluorescence intensity, and the Western blot method was used to detect the expression of Beclin 1, LC3 and P62 autophagy-related proteins. The data were analyzed by one way analysis of variance. Results: The autophagy level of ARPE-19 cells can resist light damage and reduce the apoptosis rate under 12 hours of light (F=931.53, P<0.001); after 24 hours of light, the autophagy level of cells exceeded its normal regulation range, and the apoptosis rate increased (F=1156.67, P<0.001). The difference was statistically significant. The use of the autophagy inhibitor 3MA inhibited the light-induced excessive autophagy of ARPE-19 cells, reduced the rate of apoptosis (F=2.856, P=0.027), and protected cells against light damage. Conclusions: The level of ARPE-19 autophagy changes with the amount of time to light exposure. The cell apoptosis rate increases with prolonged light exposure. Inhibiting cell autophagy can protect ARPE-19 cells from light damage.