摘要目的 观察不同能量紫外线(UV)B照射对人晶状体上皮细胞(LECs)的损伤作用及细胞线粒体中谷氧还蛋白2(Grx2)的表达变化,探讨Grx2对UVB诱导的人LECs凋亡的抑制作用.方法 对HLE-B3进行体外培养,以不同能量的UVB(0、10、30、50 mJ/cm2)(波长297 nm)分别照射培养的细胞,分别于照射后2、4、8、12和16 h在光学显微镜下观察细胞形态变化;采用细胞计数试剂盒8(CCK8)法检测各组细胞的存活率;采用TUNEL法测定各组细胞的凋亡率;分别采用实时荧光定量PCR和Western blot法检测各组细胞中Grx2 mRNA及其蛋白的相对表达量.用pcDNA3.1-Grx2质粒转染培养的细胞构建Grx2过表达模型,以pcDNA3.1空质粒转染组作为对照,并以UVB照射转染的细胞,采用TUNEL法检测各组细胞的凋亡率.结果 细胞培养过程中未照射组细胞贴壁生长,细胞伸展良好,细胞中Grx2表达呈绿色荧光.UVB照射的细胞皱缩变小,死亡细胞增多,10、30、50 mJ/cm2 UVB照射后随着UVB剂量增加和照射时间延长细胞存活率逐渐下降,凋亡细胞逐渐增多.10 mJ/cm2 UVB照射组和30 mJ/cm2 UVB照射组照射后4h,细胞中Grx2 mRNA相对表达量分别为2.53±0.48和3.53±0.14,均明显高于未照射组的1.01±0.08和1.00±0.09,50 mJ/cm2 UVB照射组照射后1h细胞中Grx2 mRNA相对表达量为15.30±3.01,明显高于未照射组的1.00±0.07,差异均有统计学意义(均P＜0.05);各照射不同时间后细胞中Grx2蛋白相对表达量变化趋势与mRNA相同.50 mJ/cm2 UVB照射后4h Grx2转染组细胞凋亡率为(15.34±1.71)％,明显低于空质粒组的(22.11±2.46)％,差异有统计学意义(t=3.189,P＜0.05). 结论 不同能量UVB照射后诱导的人LECs凋亡和损伤程度呈UVB能量依赖性和照射时间依赖性,各剂量UVB照射LECs后细胞中Grx2表达量均表现为一过性上调,Grx2表达量增加对UVB诱导人LECs凋亡有抑制作用.

abstractsObjeetive To observe the damage of human lens epithelial cells (LECs) induced by ultraviolet (UV) B radiation and the expression changes of glutaredoxin 2 (Grx2) in the cells,and to investigate the protective effects of Grx2 on human LECs against UVB-induced apoptosis.Methods Human LECs (HLE-B3) were cultured and exposed to different energy of UVB (0,10,30,50 mJ/cm2) with the wavelength of 297 nm.The morphology of the cells was examined under the optical microscope 2,4,8,12 and 16 hours after irradiation of UVB.The survival rate of the cells was evaluated with cell counting kit 8 (CCK8).The apoptosis rate of the cells was detected by TUNEL assay.Real-time quantitative PCR and Western blot were employed to detect the expressions of Grx2 mRNA and Grx2 protein in the cells,respectively.The cells were transfected with pcDNA3.1-Grx2 plasmid by lipofectamine 2000 as the Grx2 transfected group,and pcDNA3.1 plasmid was transfected into cells as the empty plasmid group.The cells were irradiated by 50 mJ/cm2 for 4 hours,and the apoptosis rate of human LECs was detected by TUNEL assay.Results Cultured cells grew well with the green fluorescence for Grx2 expression in the non-UVB exposure group,and shrinkage and death of the cells were found after UVB irradiation.The survival rate of the cells was gradually reduced after irradiation of 10,30,50 mJ/cm2 UVB as the increase of UVB energy and lapse of time.The expression level of Grx2 mRNA were 2.53±0.48 and 3.53±0.14 in the 10 mJ/cm2 UVB group and 30 mJ/cm2 UVB group 4 hours after irradiation,which were significantly higher than 1.01±0.08 and 1.00±0.09 in the non-UVB exposure group (all at P＜0.05).The expression level of Grx2 mRNA in the cells was 15.30±3.01 at 1 hour after irradiation in the 50 mJ/cm2 UVB group,which was significantly higher than 1.00 ±0.07 in the non-UVB exposure group (P＜0.05).The expression levels of Grx2 protein showed the same tendency to Grx2 mRNA.The apoptosis rate of the cells in the Grx2 transfected group was (15.34± 1.71) ％,and that in the empty plasmid group was (22.11 ± 2.46) ％ at 4 hours after 50 mJ/cm2 UVB irradiation,with a significant difference between them (t =3.189,P ＜ 0.05).Conclusions UVB irradiation induced damage of human LECs in dose-and time-dependent manner.However,the expression of Grx2 in human LECs is up-regulated transiently after exposure of UVB,which has a protective effect on the cells against the UVB-induced apoptosis.