泛素羧基末端水解酶L1在年龄相关性白内障晶状体上皮细胞中的表达及其抗氧化作用

摘要

背景 氧化损伤是年龄相关性白内障发生的主要原因,而泛素蛋白酶系统参与晶状体的分化发育,研究发现其关键酶泛素羧基末端水解酶L1( UCHL1)参与帕金森病和阿尔茨海默病等年龄相关性疾病的发生发展,且与氧化应激有关. 目的 研究UCHL1在年龄相关性白内障发病过程中的作用.方法 收集24例单纯年龄相关性白内障患者术后获得的晶状体囊膜(皮质性白内障12例、核性白内障12例)、5例正常人晶状体前囊膜上皮和人晶状体上皮细胞(LECs)系SRA01/04细胞,采用免疫荧光法检测UCHL1在各组人晶状体前囊膜上皮细胞中的表达情况.构建UCHL1真核表达质粒,鉴定后采用脂质体转染法转染SRA01/04细胞作为UCHL1过表达组,同时采用绿色荧光蛋白(GFP)真核表达质粒转染SRA01/04细胞作为GFP过表达组,使用梯度过氧化氢叔丁醇(TBHB)处理24h后,采用MTT法检测各组人LECs的活性变化.结果 免疫荧光检测表明,UCHL1在各组人LECs中均有表达,但在正常晶状体囊膜、皮质性白内障以及核性白内障晶状体囊膜上皮细胞表达量的总体差异有统计学意义(F=13.441,P=0.000).皮质性白内障组以及核性白内障组晶状体囊膜上皮细胞中UCHL1的表达量均低于正常晶状体组(P=0.000、0.000),但皮质性白内障组和核性白内障组之间UCHL1的表达量差异无统计学意义(P=0.164).Western blot鉴定结果表明,UCHL1真核表达质粒转染后可见SRA01/04细胞中UCHL1的强表达.MTT检测结果显示,0.3 mol/L TBHB处理24 h后,UCHL1过表达组细胞活性吸光度(A570/630)值与GFP过表达组比较有增高的趋势,而0.2、0.4、0.5 mol/LTBHP均导致SRA01/04细胞的耐受或者大量凋亡. 结论 UCHL1具有抗氧化作用,且可能在年龄相关性白内障的发生发展过程中起抑制作用.%Background Oxidative damage is a major cause of age-related cataracts,and the ubiquitinproteasome system is involved in lens differentiation and development.Ubiquitin carboxy-terminal hydrolase L1 (UCHL1),one of key enzymes of ubiquitin-proteasome system,was discovered to participate in the age related diseases and oxidative stress damage. Objective This study was to investigate the effects of UCHL1 on the formation and development of age-related cataract. Methods Lens capsule were collected from 24 patients with age-related cataract(including 12 cases of cortical cataract and 12 cases of nuclear cataract) during the surgery.Five normal lens capsule membranes were obtained from eye bank of Tongji University.Human lens epithelial cells (LECs) line (SRA01/04) was also collected in this study.Expression of UCHL1 in the lens epithelial layer of different samples was assayed using immunofluorescence technology.UCHL1 eukaryotic expressing vector was constructed and transfected into cultured SRA01/04 by liposome,and green fluorescent protein (GFP) eukaryotic expressing vector was transfected at the same method as the control group.UCHL1 over-expressing cells were then exposed to different concentrations (0.2,0.3,0.4 and 0.5 mol/L) of tert-butyl hydroperoxide (TBHP) for 24 hours and subsequently monitored for cell viability evaluation by MTT assay. Results Immunofluorescence showed that UCHL1 was expressed in human lens epithelial layer,but significantly different expressing levels were seen among normal lens capsular membrane,cortical cataract and nuclear cataract ( F =13.411,P =0.000),and UCHL1 expressing levels were lower in cortical cataract and nuclear cataract than the normal lens (P =0.000,P =0.000).No significant difference was found in UCHL1 expressing level between cortical cataract and nuclear cataract ( P =0.164).Western blot analysis verified that UCHL1 exhibited a stranger expression in the UCHL1 transfected group compared with the GFP transfected group,illuminating a successful transfection of UCHL1 in SRA01/04 cells.MMT assay revealed that the A570/630 value in UCHL1 transfected cells was significantly elevated in comparison with GFP transfected cells following the treatment of 0.3 mol/L TBHP. Conclusions UCHL1 has an antioxidative ability,and it might plays an important role in the progress of age-related cataract.