中国现代神经疾病杂志 ›› 2014, Vol. 14 ›› Issue (11): 972-978. doi: 10.3969/j.issn.1672-6731.2014.11.010

• 癫痫基础与临床研究 • 上一篇    下一篇

2 神经元癫痫模型miRNA-212对BDNF/TrkB 信号转导通路影响的研究

蔡浩, 吴秋静, 朱延霞, 赵文, 李斌, 宋毅军   

  1. 300052 天津医科大学总医院神经内科(蔡浩、宋毅军),保健医疗部(赵文);300070 天津医科大学七年制2007级(吴秋静);300280 天津海滨人民医院神经内科(朱延霞,李斌)
  • 出版日期:2014-11-25 发布日期:2014-11-29
  • 通讯作者: 宋毅军 (Email:songyijun2000@126.com)
  • 基金资助:

    卫计委国家临床重点专科建设项目;天津市应用基础与前沿技术研究计划项目(项目编号:14JCZDJC35400);天津市应用基础与前沿技术研究计划项目(项目编号:14JCYBJC28300);天津市滨海新区卫生局科技重点项目(项目编号:2013BWKZ003)

Study of miRNA-212 regulating BDNF/TrkB signaling pathway in epileptic neuron model

CAI Hao1, WU Qiu-jing2, ZHU Yan-xia3, ZHAO Wen4, LI Bin3, SONG Yi-jun1   

  1. 1Department of Neurology, 4Department of Geriatrics, Tianjin Medical University General Hospital, Tianjin 300052, China
    2Grade 2007, 7-year Graduate School, Tianjin Medical University, Tianjin 300070, China
    3Department of Neurology, Tianjin Haibin People's Hospital, Tianjin 300280, China
  • Online:2014-11-25 Published:2014-11-29
  • Contact: SONG Yi-jun (Email: songyijun2000@126.com)
  • Supported by:

    This study was supported by National Key Clinical Speciality Construction Project Launched by the National Health and Family Planning Commission of the People's Republic of China, Tianjin Research Program of Application Foundation and Advanced Technology (No. 14JCZDJC35400, 14JCYBJC28300) and Key Support Project of Tianjin Binhai New Area (No. 2013BWKZ003).

摘要: 目的 探讨海马神经元癫痫模型转染微小RNA(miRNA)-212 对脑源性神经营养因子(BDNF)/酪氨酸蛋白激酶B(TrkB)信号转导通路的影响。方法 经体外培养7 d 的大鼠海马神经元(正常对照组),通过无镁离子细胞外液处理(3 h)方法建立癫痫神经元模型(癫痫模型组),继而以含镁离子正常细胞外液与慢病毒稀释液转染构建miRNA-212慢病毒表达载体;免疫荧光双染法、膜片钳技术和免疫印迹法观察转染miRNA-212 对BDNF/TrkB 信号转导通路的影响。结果 无论是正常海马神经元或癫痫神经元模型,与BDNF 共培养后其磷酸化TrkB(pTrkB)/TrkB 比值均显著升高(P = 0.001),表明BDNF/TrkB 信号转导通路被激活;转染miRNA-212 后,癫痫神经元模型pTrkB/TrkB 比值降低并低于未转染者(P = 0.001),提示BDNF/TrkB 信号转导通路受到抑制。结论 BDNF 具有激活海马神经元BDNF/TrkB 信号转导通路作用,而转染miRNA-212可抑制BDNF/TrkB 信号转导通路。

关键词: 癫痫, 海马, 脑源性神经营养因子, 蛋白酪氨酸激酶类, 微RNAs, 转染, 免疫印迹法, 细胞, 培养的

Abstract: Objective  To study the effect of epileptic neuron model after transfection of microRNA (miRNA)-212 on brain-derived neurotrophic factor (BDNF)/tyrosine protein kinase B (TrkB) signaling pathway.  Methods  The primary rat hippocampal neurons were cultivated in vitro for 7 d and were randomly divided into 8 groups: control group, epilepsy group, control + BDNF group, epilepsy + BDNF group, control + miRNA-212 group, epilepsy + miRNA-212 group, control + miRNA-212 + BDNF group, epilepsy + miRNA-212 + BDNF group. Epilepsy model of hippocampal neurons were established by being exposed to Mg2+ free extracellular fluid for 3 h. And then the neurons were put back into the normal extracellular fluid of magnesium for 2 h and slow virus diluent was dropwise added for transfection, so that miRNA-212 lentiviral vector was structured. Protein was extracted after 48-72 h. BDNF was added into media 10 min before protein was extracted. Immunofluoresence double staining, patch clamp technique and Western blotting were used to observe the effect of miRNA-212 transfection on BDNF/TrkB signaling pathway. Results After BDNF was injected, compared with the control group and epilepsy group, the phosphorylated TrkB (pTrkB)/TrkB value was significantly higher in control + BDNF group and epilepsy + BDNF group (P = 0.001), suggesting the BDNF/TrkB signaling pathway was activated. After transfection of miRNA-212, the pTrkB/TrkB value in epilepsy + miRNA-212 + BDNF group was significantly lower than that in epilepsy + BDNF group (P = 0.001), suggesting the BDNF/TrkB signaling pathway was suppressed.  Conclusions  When hippocampal neurons were transfected miRNA-212, the BDNF/TrkB signaling pathway was suppressed, but BDNF could activate the BDNF/TrkB signaling pathway.

Key words: Epilepsy, Hippocampus, Brain-derived neurotrophic factor, Protein-tyrosine kinases, MicroRNAs, Transfection, Immunoblotting, Cells, cultured