高糖诱导的牛视网膜血管内皮细胞中共济失调毛细血管扩张突变基因激酶活性变化及其对细胞氧化应激状态的影响  

Influence of ataxia-telangiectasia mutated activation on cellular oxidative stress induced by high glucose in bovine retinal capillary endothelial cells

作　　者：鲁理 郑志[2] 李静文[1] 肖文玮 李春霞[3] 

机构地区：[1]蚌埠医学院,233030 [2]上海交通大学附属第一人民医院眼科 [3]上海市中西医结合医院眼科

出　　处：《中华眼底病杂志》2014年第6期604-607,共4页Chinese Journal of Ocular Fundus Diseases

摘　　要：目的 观察高糖诱导的牛视网膜血管内皮细胞（BRECs）中共济失调毛细血管扩张突变基因（ATM）激酶的活性变化及其对细胞氧化应激状态的影响.方法 体外培养BRECs,取4-8代细胞用于实验.采用含5 mmol/L葡萄糖（正常糖组）、30 mmol/L葡萄糖（高糖组）、30 mmol/L葡萄糖和10 μmol/LATM激酶特异性抑制剂KU55933（干预组）的细胞培养液培养BRECs.细胞培养48 h后,采用蛋白免疫印迹法检测细胞中磷酸化ATM（P-ATM）、磷酸化细胞分裂素活化蛋白激酶（P-P38）及磷酸化细胞外信号调节激酶（P-ERK） 1/2的蛋白表达水平;酶联免疫吸附试验检测细胞上清液中血管内皮生长因子（VEGF）的含量;细胞内活性氧（ROS）水平检测试剂盒检测细胞内ROS水平;碘化丙啶和赫斯特染料双染色法检测细胞凋亡情况;异硫氰酸荧光素标记的葡聚糖检测内皮细胞屏障的通透性.结果 与正常糖组比较,高糖组P-ATM、P-P38、P-ERK1/2蛋白表达均增高;与高糖组比较,干预组P-ATM蛋白表达降低,p-p38、p-ERK1/2蛋白表达明显增加.3组间P-ATM、P-P38、P-ERK1/2蛋白表达比较,差异均有统计学意义（F=436.00、14 010.00、985.10,P＜0.05）.与正常糖组比较,高糖组、干预组细胞上清液中VEGF含量均升高,干预组升高幅度更明显;3组间细胞上清液中VEGF含量比较,差异有统计学意义（F=278.00,P＜0.05）.高糖组ROS含量（t=2.98、10.91）、BRECs细胞凋亡率（t=4.31、11.14）均较正常糖组升高,而又明显低于干预组,差异均有统计学意义（P＜0.05）.与正常糖组比较,高糖组、干预组细胞旁通透率均升高,干预组升高更明显;3组间细胞旁通透率比较,差异有统计学意义（F=2 223.00,P＜0.05）.结论 高糖诱导的BRECs中P-ATM表达、ROS水平、细胞凋亡率及细胞旁通透率均增高,其机制可能与P38、ERK、VEGF有关.Objective To investigate the influence of Ataxia-telangiectasia mutated （ATM） activation on cellular oxidative stress induced by high glucose in bovine retinal capillary endothelial cells （BRECs）.Methods The BRECs were treated by different culture medium with various glucose concentrations （5 mmol/L glucose,30 mmol/L glucose,30 mmol/L glucose＋10 μmol/L KU55933） as normal glucose group,high glucose group and treatment group respectively.After the cells incubated for 48 hours,the protein expression of ATM,P-ATM,Mitogen-Activated Protein Kinase P38（P38）,P-P38,Extracellular signal-regulated kinases（ERKs）,P-ERKs was detected by Western blot; cellular ROS level was detected by Reactive Oxygen Species Assay Kit ; propidium iodide/Hoechst staining was used for analysis of apoptosis; the expression of vascular endothelial growth factor （VEGF） in the supernatant was determined by EnzymeLinked Immunosorbent Assay （ELISA）; the paracellular permeability between endothelium cells was detected by FITC-dextran.Results Compared with the protein level of P-ATM,P-P38 and P-ERKs in high glucose group increased.Especially,P-P38,P-ERKs expressed much more than in high glucose group.The secretion of VEGF in high glucose group was higher than that in the normal glucose group but less than that in treatment group.The same tendency existed in ROS assay,apoptosis assay and paracellular permeability measuring.Conclusions High glucose induced altered activation of ATM which might play a protective role in cellular oxidative stress.Deficiency of ATM might lead to ROS explosion,cell apoptosis and dysfunction of endothelial barrier.The mechanism might be associated with P38,ERKs and VEGF.

关 键 词：氧化性应激/免疫学 内皮细胞/酶学 动物实验 

分 类 号：R774.1[医药卫生—眼科]