MsrB1基因对H2O2诱导的人晶状体上皮细胞凋亡的保护作用  

Role of MsrB1 gene in protection human lens epithelial cells against H2O2-induced apoptosis

作　　者：贾义[1] 陈晋[1] 

机构地区：[1]贵阳医学院生物与工程学院,550025

出　　处：《中华实验眼科杂志》2015年第4期323-327,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：贵州省科学技术基金项目（黔科合J字[2014]2028号）

摘　　要：背景 氧化应激被认为是年龄相关性白内障发生的主要因素之一,研究表明蛋氨酸亚砜还原酶B1（MsrB1）对于保护晶状体细胞抵抗氧化应激具有重要作用,然而,MsrB1基因沉默对H2O2诱导的人晶状体上皮细胞（LECs）的凋亡影响的机制尚不十分清楚.目的 探讨MsrB1基因沉默对H2O2诱导的人LECs凋亡及其细胞周期分布的影响.方法人LECs细胞系SRA01/04在DMEM中进行培养,不同浓度的H2O2（200、400、600、800和1 000μmol/L）加入培养基分别处理细胞12、24和36 h以选择最适浓度和作用时间.细胞分为正常对照组、MsrB1 siRNA转染组（将MsrB1 siRNA Lipofectamine 2000质粒转染入LECs）、H2O2诱导组（200μmol/L作用LECs 24 h）和MsrB1 siRNA转染联合H2O2诱导组（转染MsrB1 siRNA脂质质粒后用H2O2处理细胞）.采用MTT法检测不同浓度H2O2诱导后人LECs 12、24和36 h细胞的存活率,并筛选H2O2处理作用最适的浓度和时间,用于进一步的细胞凋亡诱导实验;Real-time PCR法检测MsrB1 siRNA转染后24 h各组细胞中MsrB1 mRNA的相对表达水平;采用Hoechst33528染色法观察各组细胞的细胞核形态变化;采用碘化丙啶（PI）染色法,用流式细胞仪检测细胞凋亡率和不同细胞周期的细胞比例.结果 正常对照组、H2O2诱导组、MsrB1 siRNA转染组细胞中MsrB1 mRNA的相对表达值为1.063±0.058、1.013±0.049和0.235±0.024,MsrB1 siRNA转染组细胞中MsrB1 mRNA的相对表达值明显低于正常对照组和H2O2诱导组,差异均有统计学意义（t=31.744、35.807,均P＜0.001）.不同浓度H2O2处理后24 h细胞活力最低,以200 μmol/LH2O2处理后24 h为诱导人LECs凋亡的浓度和时间点.Hoechst 33528染色显示,H2O2诱导组可见部分细胞核收缩,MsrB1 siRNA转染组可见少数收缩变小的细胞核,MsrB1 siRNA转染联合H2O2诱导组收缩变小的细胞核多于H2O2诱导组.流式细胞术检测结果显示,正常对照组的凋亡细胞占（1.36±0.35）％,而MsrB1siRNA转�Background Oxidative stress is one of the major risk factors for age-related cataract,methionine sulfoxide reductase B1 （MsrB1） has been shown to play an important role for lens cell function,resistance to oxidative stress.However,the mechanism of MsrB1 gene silencing on H2O2-induced human lens epithelial cells （LECs）apoptosis remains virtually unknown.Objective This study attempted to investigate the role of MsrB1 gene in protecting human LECs against H2O2-induced damage.Methods Human LECs cell line SRA01/04 cells were cultured in DMEM medium.Different concentrations （200,400,600,800 and 1 000 μmol/L） of H2O2 were added to medium treat the cells for 12,24 and 36 hours respectively to select the optimum inducing dose and time.The cells were divided into 4 groups.Lipofectamine 2000 containing MsrB1 siRNA was transfected into the cells in the MsrB1 siRNA transfected group,200 μmol/L H2O2 treated the cells for 24 hours as the H2O2-induced group,and MsrB1siRNA was transfected into the cells before H2O2 treatment as the MsrB1 siRNA transfected combined H2O2-induced group.The routinely cultured cells were as normal control group.Cell viability was detected by MTT assay.The relative expression of MsrB1 mRNA in the cells was analyzed by real-time PCR.Morphological changes of nuclei in the cells of different groups were observed using Hoechst 33528 staining under the fluorescence microscope.Cell apoptosis and cell cycle were quantified using flow cytometry.Results The relative expressing values of MsrB1 mRNA were 1.063±0.058,1.013 ±0.049 and 0.235 ±0.024,and the expressing values of MsrB1 mRNA in the MsrB1 siRNA transfected group were significantly lower than those of the normal control group and H2 O2-induced group （t =31.744,35.807,both at P＜O.001）.The cell viability was lowest 24 hours after 200 μmol/L H2O2 induced.Hoechst 33528 staining showed less contractive decrescent nuclei in the MsrB1 siRNA transfected group,and those in the H2 O2-induced group and MsrB1 siRNA transfected combined H2 O2-i

关 键 词：蛋氨酸亚砜还原酶 基因沉默/药物作用 人 晶状体 上皮细胞/药物作用 细胞凋亡 细胞周期 

分 类 号：R776.1[医药卫生—眼科]