不同年龄段人视网膜色素上皮细胞氧化应激过程中一氧化氮的水平分析

目的　探讨不同年龄段人视网膜色素上皮（retinal pigment epithelium，RPE）细胞氧化应激过程中一氧化氮（nitric oxide，NO）的释放水平以及色素上皮衍生因子（pigment epithelium-deriverd factor，PEDF）对不同年龄段人RPE细胞中NO释放的影响。方法　将原代RPE细胞（分别来源于9～20岁，50～55岁，60～70岁，>70岁人群）用含体积分数10%胎牛血清的DMEM/F12培养基培养，分为四组进行实验，分别为不加任何物质干预的阴性对照组，单独加300 μmol·L^-1 H₂O₂ 氧化损伤组，单独加250 μg·L^-1 PEDF处理保护组以及同时加入250 μg·L^-1 PEDF+300 μmol·L^-1 H₂O₂的损伤保护组，处理12 h，随后用NO检测试剂盒对各组细胞NO水平进行检测。结果　倒置相差显微镜观察培养的人原代RPE细胞内可见清晰透明的圆形细胞核以及双核，胞浆内含丰富黑色素颗粒。H₂O₂氧化损伤组各年龄段细胞相邻细胞发生粘连，细胞皱缩加重，死亡及悬浮细胞增多。PEDF处理保护组较H₂O₂氧化损伤组中的细胞胞质更加饱满，死亡细胞数量显著减少。随着年龄的增长，未作任何处理的50～55岁、60～70岁、>70岁RPE细胞内NO的释放水平分别是9～20岁年龄段的1.37倍、1.25倍、1.62倍，差异均无统计学意义（均为P>0.05）。随着年龄的增加，H₂O₂氧化损伤组9～20岁、50～55岁、60～70岁，>70岁RPE细胞内NO的释放水平分别是阴性对照组的1.35倍、1.15倍、0.98倍及1.05倍，差异均无统计学意义（均为P>0.05），而PEDF处理保护组能够保护氧化损伤条件下不同年龄段人RPE细胞，但PEDF处理保护组9～20岁、50～55岁、60～70岁、>70岁RPE细胞内NO的释放水平分别是阴性对照组的1.17倍、1.10倍、1.04倍及0.96倍，NO的释放量差异均无统计学意义（均为P>0.05）。结论　在不同年龄段人RPE细胞中NO的两种完全相反作用可能处于一个动态平衡中，故NO释放水平无明显变化。PEDF对RPE细胞的保护作用不通过NO途径。

Nitric oxide level in retinal pigment epithelium cells derived from groups with different ages under oxidative stress

Objective　To investigate the release of nitric oxide（NO） in retinal pigment epithelium （RPE） cells under oxidative stress and the effects of pigment epithelium-derived factor （PEDF） on NO release in different age RPE cells.Methods　Primary RPE cells （the donor ages from 9-20 years，50-55 years，60-70 years and more than 70 years） were cultured in DMEM/F12 containing 10% fetal bovine serum.The obtained RPE cells were divided into four groups:the normal control group，the oxidative damage group which were treated by 300 μmol·L^-1 hydrogen peroxide（H₂O₂），the PEDF protection group which were treated with 250 μg·L^-1 PEDF，the damage-protective group which were treated with 300 μmol·L^-1 H₂O₂ and 250 μg·L^-1 PEDF for 12 h，respectively.NO levels were detected by using NO detection kit.Results　The clear and transparent round nuclei and binuclear cells were observed by using phase contrast microscope，with the rich melanin particles in the cytoplasm.The cells in the oxidative damage group were shrinking aggravatingly and cell death increased，while the number of these cells was increased after PEDF treatment.We also discovered the shrink aged cells and the numbers of cell death were reduced in the PEDF protection group.With increasing age，the levels of NO release in cells derived from group aged 50-55 years，60-70 years and more than 70 years were 1.37 folds，1.25 folds，and 1.62 folds than that of 9-20 years old in normal RPE cells.There was no significant difference of NO levels in RPE cells with age （all P>0.05）.With increasing age，the release level of NO in the cells derived from people aged 9-20 years，50-55 years，60-70 years and more than 70 years of the oxidative damage group were 1.35 folds，1.15 folds，0.98 fold and 1.05 folds than those of the normal control group.There was also no significant difference in release levels of NO （all P>0.05）.PEDF could protected RPE cells in different age groups under oxidative stress，however，with increasing age，the release level of NO in the cells derived from people aged 9-20 years，50-55 years，60-70 years and more than 70 years of the PEDF protection group were 1.17 folds，1.10 folds，1.04 folds and 0.96 folds than those of the normal control group，and no significant difference was found in release levels of NO （all P>0.05）.Conclusion　The two diametrically opposite effects of NO in RPE cells derived from groups with different ages may be in a dynamic equilibrium，which leads to no significant change on the NO release.The protective effect of PEDF on RPE cells is not related to the NO pathway.