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