高压氧对体外培养人视网膜色素上皮细胞氧化损伤的影响  

Effect of hyperbaric oxygen on oxidative damage in human retinal pigment epithelium cells cultured in vitro

作　　者：李佳霖 赵怡臻 栾洁[1] Li Jialin;Zhao Yizhen;Luan Jie(Department of Ophthalmology,Zhongda Hospital,Southeast University,Nanjing 210009,China)

机构地区：[1]东南大学附属中大医院眼科,南京210009 [2]南京明基医院

出　　处：《中华航海医学与高气压医学杂志》2021年第5期628-633,645,共7页Chinese Journal of Nautical Medicine and Hyperbaric Medicine

摘　　要：目的探讨高压氧(HBO)干预对体外培养的正常和缺氧状态人视网膜色素上皮(RPE)细胞氧化损伤的影响。方法体外培养正常和缺氧状态(200μmol/L CoCl_(2)干预)人RPE-19细胞,随机分成对照组(正常对照组和缺氧对照组)和HBO组[正常HBO组(0.15 MPa HBO组,0.20 MPa HBO组,0.25 MPa HBO组)和缺氧HBO组(缺氧+0.15 MPa HBO组,缺氧+0.20 MPa HBO组,缺氧+0.25 MPa HBO组)]。各HBO组在纯氧下分别暴露60、90 min,每隔24 h暴露一次,共暴露2次。以MTT法检测各组RPE细胞的增殖活力;以免疫细胞化学方法检测RPE细胞内8-羟基脱氧鸟苷酸(8-OHdG)的表达量;以Western blotting法检测RPE细胞内人8-羟基鸟嘌呤糖苷酶(hOGG1)的表达量。每组实验均重复3次。结果在HBO干预60、90 min后,对于正常RPE细胞,与正常对照组相比,各压力HBO干预组细胞OD570值均降低(P<0.01),且随着压力的升高而降低(P<0.05);细胞内8-OHdG表达量明显增加(P<0.01),且随着压力的升高而增加(P<0.05);细胞内hOGGl表达量增加(P<0.01),且在60min组随着压力的升高而降低(P<0.05)。对于缺氧RPE细胞,与缺氧对照组相比,各压力HBO干预组细胞OD570值均升高(P<0.01),且在60 min组随着压力的升高而升高(P<0.01);细胞内8-OHdG表达量明显降低(60 min P<0.01,90 min P<0.05),且60 min组随着压力的升高而降低(P<0.05);细胞内hOGGl表达量明显增加(P<0.01),且随着压力的升高而增加(60 min P<0.05,90 min P<0.01)。结论HBO暴露可导致正常RPE细胞的氧化损伤,但可修复缺氧导致的RPE细胞的氧化损伤。Objective To investigate the effect of hyperbaric oxygen(HBO)intervention on oxidative damage in human retinal pigment epithelium(RPE)cells cultured in vitro in normoxic and hypoxic states.Methods The ARPE-19 cells cultured in vitro in normoxic state and the ARPE-19 cells cultured in vitro in hypoxic state(incubated in 200μmol/L CoCl_(2))were randomly divided into control groups(normoxic control group and hypoxic control group)and HBO groups[normoxic HBO group(0.15 MPa HBO group,0.20 MPa HBO group,0.25 MPa HBO group)and hypoxic HBO groups(hypoxic state+0.15 MPa HBO group,hypoxic state+0.20 MPa HBO group,hypoxic state+0.25 MPa HBO group)].Each HBO group was exposed to pure oxygen for 60 and 90 min,respectively,at 24 h intervals,for a total of two exposures.The proliferation viability of RPE cells in each group was measured by microculture tetrazolium assay(MTA);the expression level of 8-hydroxydeoxyguanosine(8-OHdG)in RPE cells was measured by immunocytochemistry(ICC);and the expression level of human 8-hydroxyguanine glycosidase(hOGG1)in RPE cells was measured by Western blotting.Each set of experiments was repeated three times.Results After 60 min and 90 min of HBO intervention,compared with the normoxic control group,the optical density 570(OD570)values of normoxic RPE cells in each normoxic HBO group were decreased(P<0.01),and continued to decrease with the increase of HBO pressure(P<0.05);the expression levels of 8-OHdG were increased significantly(P<0.01),and continued to increase with the increase of HBO pressure(P<0.05);the expression levels of hOGG1 were increased(P<0.01),and those in the HBO intervention groups after 60 min HBO intervention were decreased with the increase of HBO pressure(P<0.05).Compared with the hypoxic control group,the OD570 values of hypoxic RPE cells in each hypoxic HBO group were increased(P<0.01),and those in the HBO intervention groups after 60 min HBO intervention were increased with the increase of HBO pressure(P<0.01);the expression levels of 8-OHdG were decreased significan

关 键 词：高压氧 视网膜色素上皮细胞 氧化损伤 8-羟基脱氧鸟苷酸 8-羟基鸟嘌呤糖苷酶 

分 类 号：R774.1[医药卫生—眼科]