| Objective: This study is aimed to establish the A2 E and blue light induced RPE cells damage model to explore the regularity of distribution of A2 E and ROS during the process of blue light induced RPE cells damage by detecting the A2 E flourescence intensity of RPE cells in cytoplasm and lysosome and the ROS concentration of RPE cells in cytoplasm,and lays the foundation for following experiment.Methods:The fourth to sixth generation of human RPE cells in vitro were divided into five groups randomly:control group,blue light alone group,A2 E load group,blue light +A2E load group and blue light + A2 E load +nifedipine group.(1)The concentration of reactive oxygen species(ROS)in cytoplasm by DCFH-DA staining was assayed by flow cytometry.(2)The flourescence intensity of A2 E in cytoplasm was assayed by flow cytometry.(3)The flourescence intensity of A2 E in lysosome by Lyso-Tracker Red DND-99 staining was assayed by flow cytometry and confocal laser scanning microscope.Results: The concentration of ROS in cytoplasm of control group(8438.70±148.06),blue light group(22803.90±3301.88),A2 E load group(14139.33±2092.67),A2 E load +blue light group(37991.23±1950.92)A2E load +blue light+ nifedipine group(32434.73±2512.59)were detected by flow cytometry,and the difference was significant among groups(F=89.123,P=0.000).The ROS concentration of RPE cells in cytoplasm of blue light alone group,A2 E load group,A2 E load + blue light group and A2 E load + blue light +nifedipine group were higher than control group,the differences were significant(P=0.000,0.011,0.000,0.000<0.05);blue light alone group,A2 E load +blue light group and A2 E load + blue light +nifedipine group were higher than A2 E load group,the differences were significant(P=0.001,0.000,0.000<0.05);A2E load + blue light group and A2 E load + blue light +nifedipine group were higher than blue light alone group,the differences were significant(P=0.000,0.000<0.05);Compared with A2 E load+ blue light group and A2 E load + blue light +nifedipine group,the A2 E load + blue light group was higher,the difference was significant(P=0.013<0.05);The flourescence intensity of A2 E in cytoplasm of control group(1595.17±23.91),blue light group(3676.37±93.90),A2 E load group(1880.07±27.31),A2 E load +blue light group(4232.87±35.52)A2E load +blue light+ nifedipine group(3979.40±17.17)were detected by flow cytometry,and the difference was significant among groups(F=1968.88,P=0.000).The A2 E flourescence intensity of RPE cells in cytoplasm of blue light alone group,A2 E load group,A2 E load + blue light group and A2 E load + blue light +nifedipine group were higher than control group,the differences were significant(P=0.000,0.011,0.000,0.000<0.05);blue light alone group,A2 E load +blue light group and A2 E load + blue light +nifedipine group were higher than A2 E load group,the differences were significant(P=0.000,0.000,0.000<0.05);A2E load + blue light group and A2 E load + blue light +nifedipine group were higher than blue light alone group,the differences were significant(P=0.000,0.000<0.05);Compared with A2 E load+ blue light group and A2 E load + blue light +nifedipine group,the A2 E load + blue light group was higher,the difference was significant(P=0.000<0.05);The flourescence intensity of A2 E in lysosome of control group(1800.80±80.39),blue light group(2117.40±26.34),A2 E load group(12129.00±38.86),A2 E load +blue light group(3635.33±0.67)A2E load +blue light+ nifedipine group(3516.83±8.61)were detected by flow cytometry,and the difference was significant among groups(F=31210.829,P=0.000).The A2 E flourescence intensity of RPE cells in lysosome of blue light alone group,A2 E load group,A2 E load + blue light group and A2 E load + blue light +nifedipine group were higher than control group,the differences were significant(P=0.000,0.000,0.000,0.000<0.05);A2E load group,A2 E load + blue light group and A2 E load + blue light +nifedipine group were higher than blue light alone group,the differences were significant(P=0.000,0.000,0.000<0.05);A2E load group and A2 E load + blue light group were higher than A2 E load + blue light +nifedipine group,the differences were significant(P=0.000,0.006<0.05);Compared with A2 E load group and A2 E load + blue light group,the A2 E load group was higher,the difference was significant(P=0.000<0.05).Conclusion:(1)Blue light and A2 E can increase the ROS concentration of RPE cells in cytoplasmand both have a synergistic effect.(2)A2E and/or blue light can damage the lysosomal membrane and increase the permeability of lysosomal membrane,thus the A2 E releasing into cytoplasm from lysosomes. |
