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目的:通过建立蓝光诱导负载A2E的RPE细胞损伤模型,探索A2E及ROS在蓝光致人RPE细胞损伤过程中的分布规律,为后续实验提供基础。方法:将在培养瓶中培养的第4-6代人RPE细胞消化,制成细胞悬液并随机分为5组,A组:对照组;B组:蓝光光照组;C组:A2E负载组;D组:A2E负载+蓝光光照组;E组:A2E负载+蓝光光照+硝苯地平组;(1)用DCFH-DA探针标记人RPE细胞,用流式细胞法检测各组细胞胞浆内ROS浓度。(2)用流式细胞法检测各组细胞胞浆内A2E荧光强度。(3)用Lyso Tracker Red标记人RPE细胞溶酶体,流式细胞法测定各组细胞溶酶体内A2E荧光强度、激光扫描共聚焦显微镜观察细胞内荧光情况。结果:(1)流式细胞法检测各组人RPE细胞胞浆内ROS浓度:对照组(8438.70±148.06)、蓝光光照组(22803.90±3301.88)、A2E负载组(14139.33±2092.67)、A2E负载+蓝光组(37991.23±1950.92)、A2E负载+蓝光+硝苯地平组(32434.73±2512.59),此差异具有统计学意义(F=89.123,P=0.000)。与对照组相比,高蓝光光照组、A2E负载组、A2E负载+蓝光组、A2E负载+蓝光+硝苯地平组胞浆内ROS浓度均高,差异有统计学意义(P=0.000,0.011,0.000,0.000);蓝光光照组、A2E负载+蓝光组、A2E负载+蓝光+硝苯地平组胞浆内ROS浓度均高于A2E负载组,差异有统计学意义(P=0.001,0.000,0.000);A2E负载+蓝光组、A2E负载+蓝光+硝苯地平组胞浆内ROS浓度均高于单纯给蓝光光照组,差异有统计学意义(P=0.000,0.000);A2E负载+蓝光组胞浆内ROS浓度高于A2E负载+蓝光+硝苯地平组,差异有统计学意义(P=0.013)。(2)流式细胞法检测各组人RPE细胞胞浆内A2E荧光强度:对照组(1595.17±23.91)、蓝光光照组(3676.37±93.90)、A2E负载组(1880.07±27.31)、A2E负载+蓝光组(4232.87±35.52)、A2E负载+蓝光+硝苯地平组(3979.40±17.17),此差异具有统计学意义(F=1968.88,P=0.000)。与对照组相比,高蓝光光照组、A2E负载组、A2E负载+蓝光组、A2E负载+蓝光+硝苯地平组胞浆内A2E荧光强度均高,差异有统计学意义(P=0.000,0.000,0.000,0.000);蓝光光照组、A2E负载+蓝光组、A2E负载+蓝光+硝苯地平组胞浆内A2E荧光强度均高于A2E负载组,差异有统计学意义(P=0.000,0.000,0.000);A2E负载+蓝光组、A2E负载+蓝光+硝苯地平组胞浆内A2E荧光强度均高于蓝光光照组,差异有统计学意义(P=0.000,0.000);A2E负载+蓝光组胞浆内A2E荧光强度高于A2E负载+蓝光+硝苯地平组,差异有统计学意义(P=0.000)。(3)流式细胞法检测各组人RPE细胞溶酶体内A2E荧光强度:对照组(1800.80±80.39)、蓝光光照组(2117.40±26.34)、A2E负载组(12129.00±38.86)、A2E负载+蓝光组(3635.33±0.67)、A2E负载+蓝光+硝苯地平组(3516.83±8.61),此差异具有统计学意义(F=31210.829,P=0.000)。与对照组相比,高蓝光光照组、A2E负载组、A2E负载+蓝光组、A2E负载+蓝光+硝苯地平组溶酶体内A2E荧光强度均高,差异有统计学意义(P=0.000,0.000,0.000,0.000);A2E负载组、A2E负载+蓝光组、A2E负载+蓝光+硝苯地平组溶酶体内A2E荧光强度均高于蓝光光照组,差异有统计学意义(P=0.000,0.000,0.000);A2E负载组、A2E负载+蓝光组溶酶体内A2E荧光强度均高于A2E负载+蓝光+硝苯地平组,差异有统计学意义(P=0.000,0.006);A2E负载组溶酶体内A2E荧光强度高于A2E负载+蓝光组,差异有统计学意义(P=0.000)。(4)激光扫描共聚焦显微镜观察A2E负载于RPE细胞后溶酶体内荧光显示:A2E产生的绿色荧光与溶酶体标记红色荧光大部分重合,可以初步说明产生的A2E大部分聚集在溶酶体内。结论:(1)蓝光光照、A2E负载可致人RPE细胞胞浆内ROS增加,且两者具有协同作用。(2)A2E和(或)蓝光照射可损伤溶酶体膜,使溶酶体膜通透性增加、破裂,从而使A2E可从溶酶体释放入胞浆。