摘要目的 本研究旨在探讨 UHRF1 调节晶状体上皮细胞抗氧化、抗凋亡的能力以及其潜在分子机制.方法 应用实时定量 PCR 检测白内障体内体外模型中 UHRF1 的表达;将不同浓度的 H2 O2 分别加入人晶状体上皮细胞中,通过 CCK-8 检测 H2 O2 对于晶状体细胞的凋亡影响.通过活性氧试剂盒检测过表达 UHRF1 后,分析对于氧化应激诱导的 HLEC-3 细胞活性氧的影响.通过免疫蛋白印记方法检测氧化应激诱导的 HLEC-3 转染过表达 UHRF1后凋亡相关蛋白 Bax 、 Bcl-2 、 Rb 和 p16INK4A蛋白的变化, caspase-3 活性试剂盒检测预转染UHRF1 后由氧化应激( 1 0 0 μmol/L ,1 2 h )诱导 HLEC-3 细胞中 Caspase-3 活性变化.结果白内障患者前囊膜以及体外模型中,UHRF1 的表达有显著降低( P =0. 032).过表达 UHRF1后能够明显逆转由氧化应激诱导的 HLEC-3 细胞活性降低以及细胞内活性因子 SOD 以及GSH-Px 降低,以及 ROS 与 MDA 表达升高.而过表达 UHRF1 3 6 h 后, H2 O2 ( 1 0 0 μmol/L )刺激1 2 h ,蛋白印迹结果显示相对于正常对照组, UHRF1 组中 Bax 、 PCNA 蛋白表达明显升高,而Bcl-2 表达明显降低. Caspase-3 活性相对于对照组,实验组也明显升高( P =0 . 0 2 1 ) .此外,相对于对照组,Rb (P =0. 041)以及 p16INK4A(P =0. 038)蛋白在过表达 UHRF1 组明显升高.结论 过表达 UHRF1 通过抑制 ROS/Rb/p16INK4A通路逆转由氧化应激诱导的 HLECs 的细胞凋亡. UHRF1 将来可能成为一种治疗白内障潜在治疗靶点.

abstractsObjective This study aims to investigate a role of UHRF1 in modulating anti-oxida-tive stress and anti-apoptosis in lens epithelial cells ( LECs ) , and elucidate its potential mechanisms . Methods Real-time quantitative PCR was performed to detect the expression level of UHRF1 in cataract patients both in vivo and vitro . HLEC-3 cell viability was test using CCK-8 activity kit by adding H2 O2 ( 1 0 0 μm , 1 2 h ) to lens epithelial cells The changes of oxidative stress-induced apoptosis-related pro-teinsBax , Bcl-2 , PCNA , Rb and p1 6INK4 A in HLEC-3 transfected with UHRF1 were detected by West-ern Blotting . The Caspase-3 activity Kit was used to detect the changes of caspase-3 activity in HLEC-3 cells induced by oxidative stress ( 1 0 0 μm , 1 2 h ) after overexpressing UHRF1 for 4 8 h . Results The expression of UHRF1 was significantly down-regulated in cataract patients both in vitro and vivo ( P =0 . 0 3 2 ) . Levels of ROS and MDA were enhanced in H2 O2 group compared with control group , while SOD and GSH-Px were markedly reduced . However , overexpression of UHRF1 significantly decreased ex-prerssions of ROS and MDA and increased the activities of SOD and GSH-Px . Overexpression of UHRF1 can significantly reversed the decrease in HLEC-3 cell activity induced by H2 O2 . After overexpressing UHRF1 for 3 6 h and then treat with H2 O2 1 0 0 μmol/L for 1 2 hours , the results of Western blot showed that the protein level of Bax and PCNA in UHRF1 group was significantly higher than that in the control group , while the expression of Bcl-2 was significantly reduced . Caspase-3 activity was also higher in the experimental group than that in the H2 O2 group after overexpressing UHRF1 ( P =0 . 0 2 1 ) . The expres-sion of Rb ( P = 0 . 041 ) and p16INK4A ( P = 0 . 03 8 ) was up-regulated by H2 O2 in the experimental group than that in the control . Conclusion UHRF1 protects HLECs against oxidative stress-induced ap-optosis via the inhibition of the ROS/Rb/p16INK4A pathway . UHRF1 could potentially become a biomarker for preventing ocular aging and cataract in the future .