氯通道阻断剂对氧化应激诱导晶状体上皮细胞凋亡影响  

Role of chloride channel inhibitors in oxidative stress-induced lens epithelial cell apoptosis

作　　者：庄晓东[1] 陈水花[1] 翁景宁[1] 

机构地区：[1]福建医科大学附属协和医院眼科,福州350000

出　　处：《中国实用眼科杂志》2014年第8期1028-1033,共6页Chinese Journal of Practical Ophthalmology

摘　　要：目的 观察氯通道阻断剂5-硝基-2 （3-苯丙胺）苯甲酸（NPPB）和二异硫氰基芪-2,2’-二磺酸（DIDS）对氧化应激诱导的晶状体上皮细胞（HLE-B3）凋亡的影响,初步探讨其抑制细胞凋亡的机制.方法 实验研究.于2010年11月至2011年6月在福建医科大学附属协和医院实验室以晶状体上皮细胞HLE B-3为实验对象,用500 μmol/L叔丁基过氧化氢（t-BHP）诱导细胞氧化应激损伤作为t-BHP处理组,常规培养的细胞作为对照组.在t-BHP存在或不存在下分别加入5、100、200 μmol/L的氯通道阻断剂NPPB或DIDS培养12 h,通过MTT法检测各组细胞存活率;Annexin V-FITC/PI双染法检测细胞凋亡率;AO/EB双荧光染色观察细胞凋亡形态学改变;采用分光光度法检测细胞中丙二醛（MDA）、三磷酸腺苷（ATP）的含量及超氧化物歧化酶（SOD）; Fluo-3/AM荧光探针检测细胞内钙离子含量的变化.结果 与空白对照组相比,t-BHP处理组细胞存活率及凋亡率降低、抗氧化酶SOD活性和细胞内ATP含量降低、细胞内MDA及钙离子含量升高,差异比较有统计学意义（P＜0.01）;与t-BHP处理组比较,NPPB或DIDS处理组（100、200 μmol/L）皆可使得细胞存活率增高及凋亡率降低,并明显减少氧化应激引起的凋亡形态,增强SOD活性及提高ATP含量,降低细胞内MDA及钙离子含量,差异比较均有统计学意义（P＜0.01）.结论 氯通道阻断剂NPPB和DIDS能抑制氧化应激诱导的HLE-B3细胞凋亡,减轻细胞的损伤.其机制可能与降低晶状体上皮细胞内钙负载及改善其氧化还原稳态的失衡有关.Objective To observe the effect of chloride channels in cell damage induced by oxidative stress and to probe into its possible mechanisms.Methods 500 μmol/L tert-butyl hydroperoxide （t-BHP） was used to induce HLE B-3 cells apoptosis,the cells treated by only t-BHP were used as t-BHP-induced group,and the cells cultured by regular method were used as control group,chloride channel blockers （NPPB or DIDS） with the concentrations of 5,100 and 200 μmol/L were added into the medium for 12h respectively and with or without 500 μmol/L t-BHP-induced the cells at the same time to induce the apoptosis in experimental groups.MTT method was used to observe the cell viability.Apoptosis was measured via the Annexin V-FITC and PI staining,the intracellular free Ca2＋ concentration was determined by Fluo-3 fluorometry.AO/EB double fluorescent staining was observed under the fluorescent microscope,and the cells were collected respectively after different treatments for measuring MDA level and SOD with the corresponding detection kit according to the manufactures instructions.Results Compared with t-BHP-induced group,apoptosis rate and the intracellular free Ca2＋ concentration in culture medium decreased after the treatment of NPPB or DIDS （100 and 200μmol/L）,cell viability in these group were significantly higher than ones of t-BHP-induced group （P 〈0.01）.Chloride channel blockers relieved cell injury caused by t-BHP,MDA content were lower than those in t-BHP-induced group （P 〈0.01）,and SOD activity and ATP content were higher than that in t-BHP-induced group （P 〈0.01）.Conclusions Blockage of chloride channels by DIDS and NPPB rescued HLE B-3 cells from t-BHP-induced apoptosis,the mechanism might be related to inhibiting the calcium overload and improve the imbalance of redox homeostasis.

关 键 词：凋亡 氯通道 细胞容积 晶状体上皮细胞 阻滞剂 

分 类 号：R776.102[医药卫生—眼科]