摘要
目的探讨体外癫痫模型海马神经元中线粒体衔接蛋白Miro1表达的改变及其意义。方法原代培养新生24 h内的SD大鼠海马神经元,通过无镁细胞外液诱导体外癫痫模型。培养至14 d的海马神经元随机分为对照组(CON组)、无镁诱导组(AE组)、AE+对照腺病毒组(AE+r Ad组)、AE+sh RNA腺病毒组(AE+r Ad-Miro1-sh RNA组)。CON组及AE组分别用正常细胞外液和无镁细胞外液作用3 h后,更换为正常维持液。AE+r Ad组、AE+r Ad-Miro1-sh RNA组分别于无镁诱导48 h前转染对照腺病毒、靶向大鼠Miro1基因的sh RNA重组腺病毒真核表达质粒。各组分别于造模后24 h终止细胞培养。采用Western blot法检测细胞内Miro1、ND6蛋白的表达,免疫荧光法检测细胞内ND6蛋白的表达,比色法测定丙二醛(MDA)、还原型谷胱甘肽(GSH)的蛋白浓度。结果与CON组比较,AE组、AE+r Adv组大鼠海马神经元Miro1表达明显升高,AE+r Ad-Miro1-sh RNA组大鼠海马神经元Miro1表达明显降低(均P<0.05);AE组、AE+r Adv组、AE+r Ad-Miro1-sh RNA组大鼠海马神经元ND6表达及GSH蛋白浓度明显降低,MDA蛋白浓度明显升高(均P<0.05)。AE+r Ad-Miro1-sh RNA组大鼠海马神经元Miro1、ND6表达及GSH蛋白浓度明显低于AE组和AE+r Adv组,MDA蛋白浓度明显高于AE组和AE+r Adv组(均P<0.05)。结论线粒体衔接蛋白Miro1可能对无镁致痫海马神经元有保护作用。抑制线粒体Miro1蛋白表达后,ND6、GSH蛋白表达减少,MDA蛋白表达增加,加重线粒体氧化应激损伤。
Objective To investigate the change and its significance of the expression of mitochondrial cohesion protein Mirol in hippocampal neurons of extracorporeal epilepsy model. Methods Newly born 24 h the SD rat hippocampal neurons was primitively cultured. Extracorporeal epilepsy model was established by Mg2＋ -free media in vitro. Hippocampa] neurons were cultured to the 14 d, and then were randomly divided into control group （CON group）, Mg2＋-free media induced group （AE group）, AE ＋ control adenovirus group （AE ＋ tAd group）, AE ＋ ShRNA targeting rats Mirol gene recombinant adenovirus group （AE ＋ rAd-Mirol-shRNA group）. CON group and AE group were cultured with normal extracellular fluid and media without magnesium respectively, then changed to maintenance media after 3 h. AE ＋ rAd group and AE ＋ rAd-Mirol -shRNA group were transfected adenovirus before induced in 48 h. Primary culture was terminated after epilepsy model builded in 24 h. The expression of Mirol and ND6 were detected by Western blot method. The expression of ND6 was detected by immunofluorescence method. The protein concentration of MDA and GSH were detected by colorimetric method. Results Compared with CON group, the expressions of Mirol was significantly increased in AE group and AE ＋ rAdv group and was significantly decreased in AE ＋ rAd-Mirol-shRNA group （ all P 〈0. 05 ） , the expression of ND6 and the protein concentration of GSH were significantly decreased, while the protein concentration of MDA was significantly increased in AE group, AE ＋ rAdv group and AE ＋ rAd-Mirol-shRNA group （ all P 〈 0. 05 ）. The expressions of Mirol, ND6 and the protein concentration of GSH in AE ＋ rAd-Mirol-shRNA group were significantly lower and the protein concentration of MDA was significantly higher than those in AE group and AE ＋ rAdv group （ all P 〈 0. 05 ）. Conclusions Mitochondrial cohesion protein Miro I may have a protective effect on the hippocampal neurons of epilepsy model induced by Mg2 ＋ - free media in vitro. When the expression of mitochondrial Mirol protein is inhibited, the expression of ND6 and GSH are decreased, the expression of MDA is increased, and the oxidative stress damage is aggravated.
作者
王英
连亚军
谢南昌
高延伦
金迪
WANG Ying;LIAN Ya-jun;XIE Nan-chang(Department of Neurology, Xunxian People＇s Hospital, Hebi 456250, Chin)
出处
《临床神经病学杂志》
CAS
2018年第2期130-133,共4页
Journal of Clinical Neurology
基金
国家自然科学基金资助项目(52110754)
关键词
线粒体衔接蛋白
Miro1
癫痫
线粒体移动
氧化应激
mitochondrial cohesion protein
Mirol
epilepsy
mitochondrial movement
oxidative stress
