Effect Of Oxidative Stress On The Expression Of ΑB-crystallin In RPE Cells

Objective To investigate the effect of oxidative stress on the expression ofαB-crystallin（CRYAB）in retinal pigment epithelial（RPE）cells.Methods（1）RPE cells treated with different concentrations of H₂O₂（blank control,100μmol/L,200μmol/L,300μmol/L）were divided into 1 h group,3 h group,and 6 h group seperately.Cell viability was detected by MTT assay.The level of CRYAB protein and gene expression in RPE cells were detected by Western Blot and RT-PCR,respectively.（2）RPE cells were transfected with fluorescently labeled negative siRNA and lipofectamin2000 reagents at different concentrations,and the optimal transfection conditions were selected for subsequent experiments.Three CRYAB interfering RNAs were designed and the one with the best silencing effect was selected for subsequent experiments.（3）RPE cells transfected for 24h and treated with 200μmol/L H₂O₂ for 6h were divided into siRNA group,siRNA+H₂O₂ group,negative control group,H₂O₂ group,and blank control group seperately.The level of CRYAB gene and protein expression in RPE cells were detected by RT-PCR and Western Blot,respectively.Results（1）After 1h treated with 100μmol/L H₂O₂,the expression of CRYAB gene in RPE cells was slightly increased（P<0.05）,but the level of CRYAB protein expression in RPE cells was not changed obviously.In the 1h（200μmol/L,and 300μmol/L H₂O₂）,3h and 6h groups,the level of gene and protein expression of CRYAB in RPE cells showed the increasing tendency with the increase of H₂O₂concentration（P<0.05）.Under the same concentration of H₂O₂ stimuli,the level of gene and protein expression of CRYAB increased with the time expanded（P<0.05）.（2）The best transfection efficiency was achieved when the ratio of siRNA to Lipofectamine 2000 was 1:0.019 and the siRNA concentration was 105nM.（3）Compared with blank control group and siRNA+H₂O₂ group,the level of CRYAB gene and protein expression in H₂O₂ group had increased statistical signif icantly（P<0.01）.Compared with siRNA+H₂O₂ group and blank control group,the level of CRYAB gene and protein expression were decreased statistical signif icantly in siRNA group（P<0.01）.Conclusion（1）The expression of CRYAB in RPE cells was H₂O₂ dose-dependent and time-dependent.（2）The designed SiRNA-CRYAB-464 could effectively silence theαB-crystallin gene of RPE cells.（3）SiRNA-CRYAB could reduce the high expression of CRYAB caused by H₂O₂（low concentration and short time stimulated）.