Src-家族酪氨酸激酶特异性抑制剂对H_2O_2介导的晶状体上皮细胞Ca^(2+)内流的影响  

Effects of specific inhibitor of Src-family tyrosine kinase on calcium in flux induced by H_2O_2 in lens epithelial cells

作　　者：汪沙[1] 周健[1] 

机构地区：[1]第四军医大学西京医院眼科全军眼科研究所,西安710032

出　　处：《眼科研究》2010年第6期530-534,共5页Chinese Ophthalmic Research

基　　金：国家自然科学基金项目(30672292);教育部新世纪优秀人才支持计划(NCET2006年度)

摘　　要：目的观察Src-家族酪氨酸激酶(SFK)抑制剂PP1对H2O2诱导的晶状体上皮细胞(LECs)内游离钙离子(Ca2+)浓度变化的影响。方法用Ca2+荧光探针Fluo-3/AM负载人晶状体HLE-B3,用激光共焦显微镜观察在不同浓度H2O2刺激后细胞内Ca2+的荧光强度变化;进一步用0.1nmol/LPP1、0.3μmol/(L·min)过氧化氢酶和DMSO分别预处理细胞,观察在常规Ca2+、低Ca2+和高Ca2+培养基条件下H2O2刺激后细胞内Ca2+荧光强度的变化,评价PP1对H2O2诱导的LECs内Ca2+的作用。结果不同浓度H2O2刺激后细胞内游离Ca2+浓度升高,呈剂量依赖效应。用0.1mmol/LH2O2刺激细胞,在常规Ca2+培养基中,PP1组和过氧化氢酶组细胞内Ca2+荧光强度增长幅度与DMSO组比较分别降低(28.5±4.2)%、(33.8±3.7)%,差异均有统计学意义(q=3.73,P<0.05;q=4.21,P<0.05)。在低Ca2+培养基中,3个组细胞内Ca2+荧光强度增强均不明显;在高Ca2+培养基中,PP1组、过氧化氢酶组的荧光强度增加幅度较DMSO组分别降低(13.5±1.8)%和(21.3±2.4)%(q=5.58,P<0.01;q=7.11P<0.01)。常规Ca2+和高Ca2+培养基中,PP1组和过氧化氢酶组细胞内Ca2+荧光强度增长幅度的差异均无统计学意义(q=3.04,P>0.05;q=2.76,P>0.05)。结论 SFK特异性抑制剂PP1能有效抑制H2O2诱导的LECs的Ca2+内流,从而阻断依赖Ca2+激活的信号转导途径,阻止皮质性白内障的发生。BackgroundResearch determined that the Ca2＋ in the lens with cortical cataract is elevated,and as the second messenger of cell,Ca2＋ participates in the stress response by activating the Src-family tyrosine kinase（SFK）.Our previous study indicated that SFK inhibitor,PP1,can arrest the opacification of lens.ObjectiveThe purpose of this study was to investigate the effects of specific inhibitor of SFK on calcium influx induced by hydrogen peroxide（H2O2）in human lens epithelial cells（LECs）.MethodsThe LECs line,HLE-B3,were loaded with Fluo-3/AM and then treated with 0.01,0.1 and 1mmol/L H2O2.The HLE-B3 without H2O2 treatment served as the control.The alteration of cellular calcium was observed under the confocal laser scanning microscope.The HLE-B3 uploaded with Fluo-3/AM were employed and pretreated with 0.1nmol/L PP1,0.3 μmol/（L·min）catalase and DMSO respectively for 30minutes,and then 0.1mmol H2O2 was added into DMEM for the evaluation of intracellular Ca2＋ change.500μmol EGTA or 1mmol/L CaCl2 were used in pretreated HLE-B3 by PP1,catalase and DMSO respectively for the assay of calcium influx in cells.ResultsThe fluorescence intensity of calcium in HLE-B3 was increased with the elevation of concentration of H2O2.When cultured with only DMEM and after stimulated by 0.1mmol H2O2,the enhanced amplitudes of fluorescence intensity reduced by 28.5%±4.2% and 33.8%±3.7% respectively in the cells pretreated by PP1 or catalase compared with ones pretreated by DMSO,showing significant difference between them（q=3.73,P0.05;q=4.21,P0.05,respectively）.When cultured with DMEM combined with 500μmol EGTA,no obvious difference was found in the fluorescent intensity of calcium among PP1,catalase and DMSO pretreated cells after stimulated with H2O2（P0.05）.When cultured with DMEM combined with 1mmol/L CaCl2,the increased amplitudes of fluorescent intensity of calcium was 13.5%±1.8% in PP1group and 21.3%±2.4% in catalase group,presenting considerable differences in comparison with DMSO group（q=5.58

关 键 词：Src-家族酪氨酸激酶 过氧化氢 晶状体上皮细胞 钙离子 

分 类 号：R96[医药卫生—药理学]