中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
6期
534-537
,共4页
泛素羧基末端水解酶L1%泛素%白内障%氧化损伤%真核表达质粒
汎素羧基末耑水解酶L1%汎素%白內障%氧化損傷%真覈錶達質粒
범소최기말단수해매L1%범소%백내장%양화손상%진핵표체질립
Ubiqutin carboxyl-terminal hydrolase L1%Ubiqutin%Cataract%Oxidative stress%Eukaryotic expressing vector
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.
揹景 氧化損傷是年齡相關性白內障髮生的主要原因,而汎素蛋白酶繫統參與晶狀體的分化髮育,研究髮現其關鍵酶汎素羧基末耑水解酶L1( UCHL1)參與帕金森病和阿爾茨海默病等年齡相關性疾病的髮生髮展,且與氧化應激有關. 目的 研究UCHL1在年齡相關性白內障髮病過程中的作用.方法 收集24例單純年齡相關性白內障患者術後穫得的晶狀體囊膜(皮質性白內障12例、覈性白內障12例)、5例正常人晶狀體前囊膜上皮和人晶狀體上皮細胞(LECs)繫SRA01/04細胞,採用免疫熒光法檢測UCHL1在各組人晶狀體前囊膜上皮細胞中的錶達情況.構建UCHL1真覈錶達質粒,鑒定後採用脂質體轉染法轉染SRA01/04細胞作為UCHL1過錶達組,同時採用綠色熒光蛋白(GFP)真覈錶達質粒轉染SRA01/04細胞作為GFP過錶達組,使用梯度過氧化氫叔丁醇(TBHB)處理24h後,採用MTT法檢測各組人LECs的活性變化.結果 免疫熒光檢測錶明,UCHL1在各組人LECs中均有錶達,但在正常晶狀體囊膜、皮質性白內障以及覈性白內障晶狀體囊膜上皮細胞錶達量的總體差異有統計學意義(F=13.441,P=0.000).皮質性白內障組以及覈性白內障組晶狀體囊膜上皮細胞中UCHL1的錶達量均低于正常晶狀體組(P=0.000、0.000),但皮質性白內障組和覈性白內障組之間UCHL1的錶達量差異無統計學意義(P=0.164).Western blot鑒定結果錶明,UCHL1真覈錶達質粒轉染後可見SRA01/04細胞中UCHL1的彊錶達.MTT檢測結果顯示,0.3 mol/L TBHB處理24 h後,UCHL1過錶達組細胞活性吸光度(A570/630)值與GFP過錶達組比較有增高的趨勢,而0.2、0.4、0.5 mol/LTBHP均導緻SRA01/04細胞的耐受或者大量凋亡. 結論 UCHL1具有抗氧化作用,且可能在年齡相關性白內障的髮生髮展過程中起抑製作用.
배경 양화손상시년령상관성백내장발생적주요원인,이범소단백매계통삼여정상체적분화발육,연구발현기관건매범소최기말단수해매L1( UCHL1)삼여파금삼병화아이자해묵병등년령상관성질병적발생발전,차여양화응격유관. 목적 연구UCHL1재년령상관성백내장발병과정중적작용.방법 수집24례단순년령상관성백내장환자술후획득적정상체낭막(피질성백내장12례、핵성백내장12례)、5례정상인정상체전낭막상피화인정상체상피세포(LECs)계SRA01/04세포,채용면역형광법검측UCHL1재각조인정상체전낭막상피세포중적표체정황.구건UCHL1진핵표체질립,감정후채용지질체전염법전염SRA01/04세포작위UCHL1과표체조,동시채용록색형광단백(GFP)진핵표체질립전염SRA01/04세포작위GFP과표체조,사용제도과양화경숙정순(TBHB)처리24h후,채용MTT법검측각조인LECs적활성변화.결과 면역형광검측표명,UCHL1재각조인LECs중균유표체,단재정상정상체낭막、피질성백내장이급핵성백내장정상체낭막상피세포표체량적총체차이유통계학의의(F=13.441,P=0.000).피질성백내장조이급핵성백내장조정상체낭막상피세포중UCHL1적표체량균저우정상정상체조(P=0.000、0.000),단피질성백내장조화핵성백내장조지간UCHL1적표체량차이무통계학의의(P=0.164).Western blot감정결과표명,UCHL1진핵표체질립전염후가견SRA01/04세포중UCHL1적강표체.MTT검측결과현시,0.3 mol/L TBHB처리24 h후,UCHL1과표체조세포활성흡광도(A570/630)치여GFP과표체조비교유증고적추세,이0.2、0.4、0.5 mol/LTBHP균도치SRA01/04세포적내수혹자대량조망. 결론 UCHL1구유항양화작용,차가능재년령상관성백내장적발생발전과정중기억제작용.
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.
