葡萄糖氧化酶诱导的氧化应激抑制α4β2烟碱型乙酰胆碱受体电流  

The oxidative stress induced by glucose oxidase suppresses the current of α4β2 nicotinic acetylcholine receptor

作　　者：赵君君[1] 郑媛[1] 渠静[1] 杨慧[1] 张建亮[1] 

机构地区：[1]首都医科大学神经生物学系教育部神经变性病重点实验室北京脑重大疾病研究院,北京100069

出　　处：《基础医学与临床》2016年第5期577-580,共4页Basic and Clinical Medicine

基　　金：国家自然科学基金(31271136;31571202);北京市教委科技计划(KZ201210025020);北京市属高等学校青年拔尖人才培育计划(CIT&TCD201504087)

摘　　要：目的探究葡萄糖氧化酶(GO)引起的氧化应激对α4β2烟碱型乙酰胆碱受体(nAChR)电流的影响。方法在HEK293T细胞共转染α4和β2质粒48 h后进行实验。分为对照组(control组)、3.5 k U/L的GO处理组(GO组)、3.5 k U/L的GO与450 k U/L的过氧化氢酶(CAT)共处理组(CAT组)。使用DCFH-DA荧光探针以及激光共聚焦显微镜来检测活性氧(ROS)的产生;采用全细胞膜片钳检测α4β2 nAChR的电流变化,给予1、10和30μmol/L ACh检测受体功能,给予10μmol/L ACh检测电流变化。结果全细胞膜片钳记录到呈ACh剂量依赖性的电流;GO处理HEK293T细胞1 h后可显著地增强细胞活性氧的水平(P<0.001);对照组电流在10次给药中均保持稳定,GO组的电流在连续10次的ACh给药下逐渐降低(P<0.01);CAT组反转GO引起的α4β2 nAChR电流下降(P<0.01)。结论GO引起的氧化应激状态导致α4β2 nAChR电流逐渐下降,α4β2 nAChR电流的下降与ROS的增多密切相关。Objective To investigate the effect of oxidative stress induced by glucose oxidase （GO） on the current of α4β2 nicotinic acetylcholine receptor （nAChR）. Methods HEK293T cells were co-transfected with plasmid α4 and β2 for 48 h. Experimental groups were divided into control group, GO group and CAT group. To measure the level of reactive oxygen species （ROS）, we used confocal microscopy to detect fluorescence intensity by DCFH-DA fluorescent probe; the currents of α4β2 nAChR were detected by whole-cell patch clamp. 1 μ moL/L, 10 μmoL/L and 30 μ mol/L ACh were used to detect the receptor function. 10 μ moL/L ACh were used to detect current changes. Results The currents from α4β2 nAChR in HEK293T cells were increased in a dose-dependent manner; the treatment of 3.5 kU/L GO for 1 h significantly incresed the level of ROS in HEK293T cells （P 〈0. 001 ） ; with the consecutive application of ACh, the currents in control group were still stable after 10 times administration, whilethe GO-induced oxidative stress gradually led to the currents of α4β2 L of catalase （CAT） prevented the currents running down （P 〈 0.01 ） nAChR run-down （P 〈 0. 01 ） ; the 450 kU/ Conclusions GO-induced oxidative stress may cause α4β2 nAChR currents run down gradually. ROS plays an important role in the current run-down of α4β2 nAChR.

关 键 词：活性氧 氧化应激 烟碱型乙酰胆碱受体 

分 类 号：R741[医药卫生—神经病学与精神病学]