miR-204对年龄相关性白内障LECs的抗氧化损伤作用及其机制  

Anti-oxydative effects of miR-204 on LECs in age-related cataract

作　　者：苏现华 王晔[2] 黄钰森[1] 

机构地区：[1]青岛大学医学院山东省眼科研究所,266071 [2]山东省眼科研究所山东省眼科学重点实验室一省部共建国家重点实验室培育基地,青岛266071

出　　处：《中华实验眼科杂志》2016年第3期227-233,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81370996）;山东省自然科学基金项目（ZR2012HM009）

摘　　要：背景miRNAs是一类非编码的小分子RNA，对LECs的凋亡发挥调控作用，但miRNAs对年龄相关性白内障患者LECs中氧化损伤的生物学效应及其机制仍需进一步研究。目的探讨miR-204在体内及体外对年龄相关性白内障氧化损伤的作用及其机制。方法收集20例年龄相关性白内障患者的晶状体前囊膜和20个正常供体眼晶状体前囊膜，采用实时荧光定量PCR法检测2个组晶状体前囊膜中miR-204的表达量。对人晶状体上皮细胞系HLE—B3进行培养，在细胞培养液中添加200μmol／LH2O2建立LECs氧化损伤模型，然后将细胞分为模型对照组、miR-204拟似物组、拟似物对照组、miR-204拮抗物组、拮抗物对照组，分别在细胞培养液中添加相应的转染剂，未用H2O2处理的LECs作为正常对照组。转染后24h采用实时定量PCR法测定各组LECs中miR-204mRNA的表达以验证转染率；采用流式细胞仪测定体外各组细胞的凋亡率；采用实时荧光定量PCR和Westernblot法检测LECs中TP53INPl及p53mRNA和蛋白的相对表达量。结果年龄相关性白内障患者晶状体前囊膜中miR-204mRNA相对表达量明显低于正常人，差异有统计学意义（t=14．21，P〈0．05）。正常对照组、模型对照组、miR-204拟似物组、拟似物对照组、miR-204拮抗物组、拮抗物对照组细胞凋亡率分别为1．31±O．12、4．90±0．28、2．60±0．15、4．39±0．20、5．74±0．13和4．34±0．63，模型对照组细胞凋亡率明显高于正常对照组，差异有统计学意义（t=-20．69，P〈0．01）；miR-204拟似物组细胞凋亡率明显低于拟似物对照组，而miR-204拮抗物组细胞凋亡率明显高于拮抗物对照组，差异均有统计学意义（t=-12．20，P〈0．01；t=3．79，P〈0．05）。实时荧光定量PCR和Westernblot法检测结果显示，模型对照组细胞中TP53INP1、p53mRNA及蛋白的相对表达量明显高于正常对照组，差异均有统计学意义（mRNA�Background miRNAs are a group of non-coding small RNA molecules, and they play an important role in regulating the apoptosis of LECs. The biological effects and mechanisams of miRNAs on LECs in age- related cataract still need to be further elucidated. Objective This study was to investigate the anti-oxidative effects of miR-204 on age-related cataract in vitro. Methods The specimens of anterior lens capsules from age- related cataract patients and normal donors were collected in Shandong Eye Institution and 20 subjects for each. The expression level of miR-204 was detected and compared between cataractous eyes and normol eyes by real-time fluorescence quantitative PCR （RT-qPCR）. HLE-B3 ceils, a human LEC line, were cultured, and the oxydativestress models of LECs were established by adding 200 μmol/L Hz Oz in the medium. The models were divided into model control group, miR-20g agomir group, agomir negative control group, miR-204 antgomir group and antagomir negative control group according to the difference of tranfected agents, and normal cells served as the control group. Twenty-four hours after transfection, the expression levels of miR-204 mRNA in the cells of various groups were detected by RT-qPCR to varify the transfection rate. Apoptosis rate of the cells was assayed by flow cytometry. The relative expression levels of TP53INP1 mRNA and p53 mRNA as well as their proteins were detected by RT-qPCR and Western blot, respectively. Results The mean expression level of miR-204 was significantly lower in the anterior lens capsules of cataractous eyes than that in the normal eyes （t = 14.21 ,P〈0. 05 ）. The mean apoptosis rate of cells was 1.31± 0. 12,4.90 ± 0.28,2.60 ± 0. 15,4.59 ±0.20,5.74 ± 0. 13 and 4.34 ± 0.63 in the normal control group,model control group, miR-204 agomir group, agomir negative control group, miR-204 antgomir group and antagomir negative control group, respectively. The apoptosis rate was significantly increased in the model control group compared with the normal control g

关 键 词：微小RNA TP53INP1 P53 年龄相关性白内障 氧化损伤 

分 类 号：R776.1[医药卫生—眼科]