解剖学报

解剖学报 ›› 2016, Vol. 47 ›› Issue (4): 433-441.doi: 10.16098/j.issn.0529-1356.2016.04.001

• 神经生物学 •    下一篇

 AKT-糖原合成激酶-3β信号通路对SOD1G93A 突变N2a细胞的保护作用

陈燕春1 管英俊1* 刘永新1 周风华1 刘焕彩2 王巧真3 马晓君1 赵春艳1   

  1. 1.潍坊医学院组织学与胚胎学教研室; 2.附属医院关节外科; 3.人体解剖学教研室， 山东 潍坊 261053
  • 收稿日期:2015-10-08 修回日期:2016-03-09 出版日期:2016-08-06 发布日期:2016-08-06
  • 通讯作者: 管英俊 E-mail:guanyj@wfmc.edu.cn
  • 基金资助:

    国家自然科学基金项目;国家自然科学基金项目;山东省自然科学基金;山东省科技发展计划项目;山东省教育厅课题;山东省教育厅课题;山东省教育厅课题

Protective role of AKT-glycogen synthase kinase-3β signaling pathway in the N2a cell with SOD1G93A mutation

CHEN Yan-chun1 GUAN Ying-jun 1* LIU Yong-xin1 ZHOU Feng-hua1 LIU Huan-cai2 WANG Qiao-zhen3 MA Xiao-jun1 ZHAO Chun-yan1   

  1. 1. Histology and Embryology Department, 2. Joint Surgery Department of Affiliated Hospital, 3. Human Anatomy Department, Weifang Medical University, Shandong Weifang 261053, China
  • Received:2015-10-08 Revised:2016-03-09 Online:2016-08-06 Published:2016-08-06
  • Contact: GUAN Ying-jun E-mail:guanyj@wfmc.edu.cn

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摘要：

目的  探讨AKT-糖原合成激酶-3β(GSK-3β)信号通路对SOD1G93A 突变N2a细胞的作用及机制。 方法 选取小鼠成神经瘤细胞系N2a，分别转染pEGFP-WT-SOD1和pEGFP-G93A-SOD1质粒，应用Western blotting和免疫荧光染色方法检测AKT、GSK-3β和细胞周期蛋白D1(cyclin D1)在细胞模型中的表达变化。应用RT-PCR和Western blotting技术检测siRNA沉默AKT后GSK-3β和cyclin D1在SOD1突变N2a细胞中的表达变化，通过MTS方法，检测细胞增殖和存活的改变。 结果 与pEGFP-WT-SOD1转染的N2a细胞比较，pEGFP-G93A-SOD1转染的N2a细胞中AKT及GSK-3β总蛋白在转染后24 h和48 h表达均无明显变化，p-AKT(Ser473)、p-GSK-3β(Ser9)和cyclin D1表达均升高。免疫荧光染色结果显示，转染后24 h和48 h，p-AKT(Ser473)、p-GSK-3β(Ser9)和cyclin D1在pEGFP-G93A-SOD1转染的N2a细胞中表达均升高。应用siRNA沉默AKT后与对照组相比，在转染后48 h和72 h，AKT、p-AKT(Ser473)、GSK-3β、p-GSK-3β(Ser9)和cyclin D1蛋白均降低。MTS实验结果显示，在AKT沉默后72 h、96 h、120 h，N2a细胞增殖和活力降低。 结论 SOD1G93A突变影响N2a细胞中AKT、GSK-3β翻译后磷酸化修饰及cyclin D1的表达，AKT可能通过调控GSK-3β和cyclin D1影响SOD1 G93A突变N2a细胞的增殖和存活。

关键词: 肌萎缩侧索硬化症, 成神经瘤细胞N2a；AKT, 糖原合成激酶-3β, 细胞周期蛋白D1, 免疫印迹法, 小鼠

Abstract:

Objective To explore the role and mechanism of AKT-GSK-3β signaling pathway in the N2a cells with SOD1G93A mutation. Methods Mouse neuroblastoma cell line N2a was transfected with pEGFP-WT-SOD1 and pEGFP-G93A-SOD1 plasmids. The expressions changes of AKT, GSK-3β and cyclin D1 were detected in the cell model using RT-PCR and Western blotting technique. The expressions of GSK-3β and cyclin D1 were detected in the N2a cells with SOD1G93A mutation regulated by knockdown of AKT, and the changes of cell proliferation and survival were determined by MTS assay. Results No significant changes were observed in AKT and GSK-3β total protein in the N2a cells transfected with pEGFP-G93A-SOD1, compared with those transfected with pEGFP-WT-SOD1. However, the phospho-AKT(Ser473), phospho-GSK-3β(Ser 9) and cyclinD1 protein in the N2a cells transfected with pEGFP-G93A-SOD1 were up-regulated significantly compared with N2a cells transfected with pEGFP-WT-SOD1 at 24 hours and 48 hours after transfection. The immunouorescence staining showed that the expressions of p-AKT(Ser473)、p-GSK-3β(Ser 9) and cyclin D1 were increased in the N2a cells transfected with pEGFP-G93A-SOD1 at 24 hours and 48 hours. AKT, p-AKT(Ser473), GSK-3β, p-GSK-3β (Ser9) and cyclin D1 protein were all downregulated significantly at 48 hours and 72 hours after knockdown of AKT compared with the negative control. MTS assay showed that N2a cell proliferation and viability were significantly decreased at 72 hours, 96 hours and 120 hours after transfection. Conclusion The mutation of SOD1 affects the phosphorylated modification of AKT and GSK-3β after translation and the expression of cyclin D1 in the N2a cells. AKT may affect the proliferation and survival of N2a cells with SOD1G93A mutation by regulating the expression of GSK-3β and cyclin D1.

Key words: Amyotrophic lateral sclerosis, Neuroblastoma cell N2a, AKT, Glycogen synthase kinase-3β, Cyclin D1, Western blotting, Mouse