SH-SY5Y细胞氧糖剥夺/恢复模型中Sestrin2基因过表达对线粒体分裂的调控研究  

Regulation of Sestrin2 overexpression in mitochondrial fission in glucose and oxygen deprivation/recovery SH-SY5Y cell model

作　　者：王秀芳 赫建帅[1] 唐莹[2] 吴秀云[1] 于文刚[1] 王亚楠 王士雷[1] Wang Xiufang;He Jianshuai;Tang Ying;Wu Xiuyun;Yu Wengang;Wang Ya'nan;Wang Shilei(Department of Anesthesiology,Affiliated Hospital of Qingdao University,Qingdao 266555,China;Department of Anesthesiology,Yidu Central Hospital,Weifang 261000,China;Department of Anesthesiology,Linyi People's Hospital,Linyi 276000,China)

机构地区：[1]青岛大学附属医院麻醉科,青岛266555 [2]潍坊市益都中心医院麻醉科,261000 [3]临沂市人民医院麻醉科,276000

出　　处：《中华神经医学杂志》2021年第8期757-764,共8页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金(81771415)。

摘　　要：目的探讨人神经母细胞瘤SH-SY5Y细胞氧糖剥夺/恢复(OGD/R)模型中应激诱导蛋白Sestrin2基因过表达对线粒体分裂的调控作用及其机制。方法(1)将SH-SY5Y细胞分为正常对照组、OGD/R组、Sestrin2过表达组及空载体组,后2组细胞预先采用慢病毒感染方法分别构建Sestrin2过表达及空载体稳转细胞株,除正常对照组细胞外均给予氧糖剥夺4 h/恢复18 h造模处理。造模完成后采用细胞计数试剂-8(CCK-8)法检测各组细胞的存活率,采用Western blotting实验检测各组细胞中Sestrin2、线粒体动力相关蛋白1(Drp1)、线粒体分裂蛋白1(Fis1)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胞浆Kelch样环氧氯丙胺相关蛋白1(Keap1)、胞核核因子E2相关因子2(Nrf2)的蛋白水平,采用透射电镜观察各组细胞中线粒体的超微结构,采用免疫荧光染色检测各组细胞中Nrf2的核移位情况。(2)将Sestrin2过表达稳转SH-SY5Y细胞株分为Sestrin2过表达组、鸦胆子苦醇+Sestrin2过表达组、二甲基亚砜(DMSO)+Sestrin2过表达组,后2组细胞在造模前分别给予Keap1/Nrf2通路抑制剂鸦胆子苦醇(终浓度100 nmol/L)或DMSO溶液(终体积分数0.1%)预处理4 h,然后各组细胞均给予氧糖剥夺4 h/恢复18 h造模处理。造模完成后采用Western blotting实验检测各组细胞中胞浆Keap1、胞核Nrf2、Drp1、Fis1的蛋白水平。结果(1)与OGD/R组相比,Sestrin2过表达组细胞的存活率明显升高(61.33%±1.15%vs.81.00%±3.00%),Bcl-2/Bax值明显升高(0.467±0.006 vs.0.880±0.010),Drp1、Fis1、胞浆Keap1蛋白水平明显降低(1.089±0.033 vs.0.865±0.014;0.829±0.009 vs.0.350±0.007;0.967±0.017 vs.0.881±0.024),胞核Nrf2蛋白水平明显升高(0.627±0.025 vs.0.957±0.015),差异均有统计学意义(P<0.05);并且,Sestrin2过表达组细胞的线粒体结构明显较OGD/R组更完整,Nrf2发生了明显的核移位。(2)与Sestrin2过表达组相比,鸦胆子苦醇+Sestrin2过表达组细胞胞核Nrf2的蛋白�Objective To investigate the role of Sestrin2 overexpression in regulating mitochondrial fission and its mechanism in human neuroblastoma SH-SY5Y cell model of glucose and oxygen deprivation/recovery(OGD/R).Methods(1)SH-SY5Y cells were divided into normal control group,OGD/R group,Vector group,and Sestrin2 overexpression group;Sestrin2 overexpression or empty vector stable cell lines in the Sestrin2 overexpression group and Vector group were constructed by lentivirus infection;cells in the later 3 groups were subjected to oxygen-glucose deprivation(OGD)for 4 h followed by restoration of O2 supply for 18 h.The cell survival rate was detected by cell counting kit(CCK)-8 assay.The protein levels of Sestrin2,dynamin-related protein 1(Drp1),mitochondrial fission protein 1(Fis1),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Kelch-like ECH-related protein 1(Keap1)in the cytoplasm and nuclear factor E2-related factor(Nrf2)in the nucleus were detected by Western blotting.The mitochondria ultrastructure was observed by transmission electron microscope.The Nrf2 nuclear translocation was detected by immunofluorescence staining.(2)Cell lines with Sestrin2 overexpression were divided into Sestrin2 overexpression group,Brusatol+Sestrin2 overexpression group,and DMSO+Sestrin2 overexpression group.Cells in the Brusatol+Sestrin2 overexpression group were pretreated with normal medium containing Brusatol(Keap1/Nrf2 pathway inhibitor,final concentration:100 nmol/L)for 4 h before OGD/R;cells in the DMSO+Sestrin2 group were pretreated with normal medium containing DMSO(final volume fraction:0.1%)for 4 h before OGD/R.Cells in these groups were then subjected to OGD for 4 h followed by restoration of O2 supply for 18 h.The protein levels of Drp1,Fis1,Keap1 in the cytoplasm,and Nrf2 in the nucleus were measured by Western blotting.Results(1)As compared with those in the OGD/R group,cells in the Sestrin2 overexpression group had significantly increased survival rate(61.33%±1.15%vs.81.00%±3.00%),significantly up-regulated Bcl-

关 键 词：Sestrin2 氧糖剥夺/恢复 线粒体分裂 Kelch样环氧氯丙胺相关蛋白1/核因子E2相关因子2通路 

分 类 号：R743.3[医药卫生—神经病学与精神病学]