G93A突变的hSOD1基因对Nrf2/ARE信号通路的影响  

Influences of hSOD1 Gene with G93A Mutation on Nrf2/ARE Signaling Pathway

作　　者：亓法英[1] 王富敏[1] 于继徐[1] 车峰远[1] 赵孔波[2] 

机构地区：[1]山东省临沂市人民医院神经内科,山东临沂276003 [2]山东省临沂市人民医院急救中心,山东临沂276003

出　　处：《中国临床医学》2015年第1期19-24,共6页Chinese Journal of Clinical Medicine

基　　金：山东省自然科学基金(编号:ZR2010HM041);山东省临沂市科学技术发展计划(编号:201413006)

摘　　要：目的:探讨G93A突变的hSOD1(human Cu/Zn superoxide dismutase)基因在肌萎缩侧索硬化转基因细胞模型NSC-34细胞中对Nrf2/ARE信号通路的影响。方法:将构建好的质粒hSOD1-pcDNA3.1(-)、hSOD1-G93A-pcDNA3.1(-)和pcDNA3.1(-)转染至肌萎缩侧索硬化转基因细胞模型NSC-34细胞内,根据转染质粒的不同分为4组:正常组、空转组、野生组和突变组。通过检测细胞内脂质过氧化产物丙二醛(malondialdehyde,MDA)的含量来判断细胞氧化损伤的程度;检测线粒体膜通透性;通过Western blotting检测细胞中Nrf2、抗氧化酶的蛋白表达水平,以反映Nrf2/ARE信号通路在各组的活化程度。结果:转染hSOD1-G93A-pcDNA3.1(-)的NSC-34细胞(突变组)内氧化应激水平增高,同时线粒体通透性增加,提示线粒体受损伤;且Nrf2以及Nrf2/ARE信号通路下游效应分子血红素氧合酶-1(heme oxygenase-1,HO-1)和NAD(P)H醌氧化还原酶-1[NAD(P)H:quinone oxidoreductase 1,NQO1]的蛋白表达水平均降低(P<0.05)。突变组细胞胞浆中Nrf2表达明显减少(P<0.05),而突变组和野生组细胞核中Nrf2增多,突变组细胞更为显著(P<0.05)。结论:hSOD1基因的G93A突变使细胞Nrf2/ARE信号通路受损,降低了细胞的抗氧化能力,加重了线粒体损伤。Objective：To investigate the effect of human Cu/Zn superoxide dismutase（hSOD1）gene with G93 A mutation on Nrf2/ARE signaling pathway in NSC-34 cell,the transgenic cell model of amyotrophic lateral sclerosis（ALS）.Methods：The established plasmids,hSOD1-pcDNA3.1（-）,hSOD1-G93A-pcDNA3.1（-）,and pcDNA3.1（-）were transfected into NSC-34 cells,the transgenic cell model of amyotrophic lateral sclerosis.The models were divided into four groups according to different transfected plasmids.They were normal group,empty group,wild group and mutation group.The oxidative-stress injury was evaluated by detecting the content of intracellular malondialdehyde（MDA）,a lipid peroxidation product.The permeability of mitochondrial membrane was detected.Western blotting was used to determine the intracellular protein expression level of Nrf2 and antioxidase,so as to reveal the activation level of the Nrf2/-ARE signaling pathway in each group.Results：The level of oxidative stress and the mitochondrial permeability increased in the NSC-34 cells transfected with human hSOD1-G93ApcDNA3.1（-）gene（mutation group,P〈0.05）,which implied impairment of mitochondrias.The protein expression level of Nrf2,significantly decreased in NSC-34 cells transfected with hSOD1-G93 A gene（P〈0.05）.So were heme oxygenase-1（HO-1）and NAD（P）H：quinone oxidoreductase 1（NQO1）,downstream effector molecules of Nrf2-ARE signaling pathway.The expression of Nrf 2 in cytoplasm significantly decreased in mutation group,while Nrf2 expression in cell nucleus significantly increased（P〈0.05）in mutation group and wild group,especially in mutation group（P〈0.05）.Conclusions：The G93 A mutation of hSOD1 gene impairs Nrf2/ARE signaling pathway in ALS cell models,reduces the antioxidant ability of cells,and increase the impairment of mitochondrias.

关 键 词：肌萎缩侧索硬化 NSC-34细胞 Nrf2/ARE通路 

分 类 号：R34[医药卫生—基础医学]