MGMT在双氧水诱导的氧化损伤模型中对晶状体上皮细胞DNA损伤和凋亡的影响  

Effects of O6-methylguanine-DNA methyltransferase on DNA damage and apoptosis of lens epithelial cells in hydrogen peroxide-induced oxidative damage models

作　　者：蔡卫华 郭仁建 CAI Weihua;GUO Renjian(Department of Ophthalmology,Suzhou Kowloon Hospital,Shanghai Jiao Tong University School of Medicine,Suzhou 215028,Jiangsu Province,China;Department of Ophthalmology,Xiaoshan Economic and Technological Development Zone Hospital,Hangzhou 311200,Zhejiang Province,China)

机构地区：[1]上海交通大学医学院苏州九龙医院,江苏省苏州市215028 [2]杭州市萧山经济技术开发区医院眼科,浙江省杭州市311200

出　　处：《眼科新进展》2024年第12期955-960,共6页Recent Advances in Ophthalmology

摘　　要：目的探讨DNA修复基因MGMT在氧化损伤环境中对晶状体上皮细胞(LECs)中DNA的损伤修复能力以及凋亡影响。方法采用不同浓度H_(2)O_(2)(0、200、400、600μmol·L^(-1))处理培养的晶状体上皮细胞(LECs),确认不同浓度H_(2)O_(2)对LECs的影响,将培养的LECs分为Control组(不做处理)、H_(2)O_(2)组(加入400μmol·L^(-1) H_(2)O_(2))、H_(2)O_(2)+HA组[加入400μmol·L^(-1) H_(2)O_(2)与对照质粒(HA)]及H_(2)O_(2)+OE-MGMT组[加入400μmol·L^(-1) H_(2)O_(2)与5μL pcDNA3.1-MGMT(过表达质粒)及12μL Lipofectamine8000(脂质体转染试剂)]。CCK-8实验检测各组细胞活力;Western blot实验检测年龄相关性白内障(ARC)患者晶状体前囊膜组织(ARC组)及各组细胞蛋白(DNA损伤标志蛋白γH2A与促凋亡蛋白Bax/Bcl2)表达水平;免疫荧光检测各组细胞DNA损伤修复能力(检测DNA损伤标志物15A3),TUNEL染色实验检测各组细胞凋亡情况。结果Western blot实验检测结果显示,Control组与ARC组患者细胞中MGMT蛋白相对表达水平分别为1.000±0.002与0.597±0.001,差异有统计学意义(P<0.0001)。LECs的MGMT蛋白相对表达水平随H_(2)O_(2)浓度上升呈下降趋势。Control组、H_(2)O_(2)组、H_(2)O_(2)+HA组及H_(2)O_(2)+OE-MGMT组γH2A相对表达水平分别为1.000±0.005、1.424±0.011、1.359±0.009、0.586±0.004,Bax/Bcl2表达水平分别为1.000±0.003、2.132±0.017、1.491±0.006、0.687±0.008,与H_(2)O_(2)+HA组比较,H_(2)O_(2)+OE-MGMT组表达均降低,差异均有统计学意义(均为P<0.001)。免疫荧光检测结果显示,Control组、H_(2)O_(2)组、H_(2)O_(2)+HA组及H_(2)O_(2)+OE-MGMT组15A3相对表达水平分别为1.000±0.128、1.842±0.207、2.104±0.267、0.723±0.073,与H_(2)O_(2)组相比,H_(2)O_(2)+OE-MGMT组15A3染色荧光强度下降,差异有统计学意义(P<0.001)。TUNEL染色检测结果显示,Control组、H_(2)O_(2)组、H_(2)O_(2)+HA组及H_(2)O_(2)+OE-MGMT组细胞凋亡的荧光强度分别为1.800±0.002、13.660±0.002、18.000±0.011、2.Objective To explore the ability of the DNA-repair gene O6-methylguanine-DNA methyltransferase(MGMT)to repair DNA damage and control apoptosis of lens epithelial cells(LECs)in an oxidative damage environment.Methods The cultured LECs were treated with different concentrations of hydrogen peroxide(H_(2)O_(2))(0μmol·L^(-1),200μmol·L^(-1),400μmol·L^(-1),and 600μmol·L^(-1))to confirm the effects of different concentrations of H_(2)O_(2) on LECs.The LECs were divided into Control group(without treatment),H_(2)O_(2) group(adding 400μmol·L^(-1) H_(2)O_(2)),H_(2)O_(2)+HA group[adding 400μmol·L^(-1) H_(2)O_(2) and hemagglutinin(HA)]and H_(2)O_(2)+OE-MGMT group[adding 400μmol·L^(-1) H_(2)O_(2) and 5μL pcDNA3.1-MGMT(overexpression plasmid)and 12μL Lipofectamine8000(liposome transfection reagent)].Cell viability was detected by Cell Counting Kit-8(CCK-8).Western blot was used to detect the expression levels of proteins(DNA damage marker proteinγH2A and pro-apoptotic protein Bax/Bcl2)in anterior capsular tissue of lens of age-related cataract(ARC)patients(ARC group)and other groups.DNA damage repair ability(DNA damage marker 15A3)of LECs in each group was detected by immunofluorescence,and apoptosis in each group was detected by TUNEL staining.Results Western blot results showed that the relative protein expression level of MGMT in LECs of the Control group and the ARC group was 1.000±0.002 and 0.597±0.001,and the difference was statistically significant(P<0.0001).The relative protein expression level of MGMT in LECs decreased with the increase of H_(2)O_(2) concentration.The relative expression level ofγH2A in the Control group,H_(2)O_(2) group,H_(2)O_(2)+HA group and H_(2)O_(2)+OE-MGMT group was 1.000±0.005,1.424±0.011,1.359±0.009,and 0.586±0.004,respectively,and the expression level of Bax/Bcl2 was 1.000±0.003,2.132±0.017,1.491±0.006,and 0.687±0.008,respectively,with those expression levels in the H_(2)O_(2)+OE-MGMT group being significantly lower than the H_(2)O_(2)+HA group(all P<0.001).Immuno

关 键 词：年龄相关性白内障 氧化损伤 MGMT基因 

分 类 号：R776.1[医药卫生—眼科]