TrkB不同亚型对癫痫海马神经元BDNF/TrkB信号通路调控的研究

目的 脑源性神经营养因子(BDNF)及其酪氨酸激酶B受体（TrkB）与癫痫关系密切，该研究探讨了癫痫神经元中TrkB不同亚型对BDNF/TrkB信号通路的调控。 方法 将14组海马神经元细胞分为钙调蛋白抑制剂（N-Acetyl-L-leucyl-L-leucyl-L-norleucinal，ALLN）组和翻译抑制剂（Anisomycin）组。免疫荧光鉴定海马神经元，无镁液处理制备癫痫模型，电生理鉴定细胞痫样放电，免疫印迹技术检测癫痫模型中TrkB和磷酸化TrkB（p-TrkB）蛋白的表达变化。 结果 细胞培养至第7天时，神经元形态特征明显，形成明显的神经网络，纯度60%-70%。经无镁细胞外液处理3小时后再恢复到正常细胞外液，电生理检测到神经元持续强直高频爆发的“癫痫样”自发性放电。免疫印迹结果显示：1. 正常+BDNF组高于正常组；2. 癫痫+BDNF组高于癫痫组，低于正常+BDNF组；3. 癫痫+ALLN+BDNF组低于癫痫+BDNF组；4. 癫痫+ALLN+BDNF组和癫痫+ALLN组差异无统计学意义;5. 癫痫+Anisomycin+BDNF组高于癫痫+BDNF组和癫痫+Anisomycin组。 结论 BDNF可激活BDNF/TrkB信号通路。癫痫状态下BDNF/TrkB通路处于抑制状态，TrkB.T的上调引起BDNF/TrkB通路的抑制，TrkB.FL表达变化不能活化BDNF/TrkB通路。

Study of the regulation of BDNF/TrkB signal pathway in hippocampal neurons by different isoforms of TrkB

Objective Brain derived neurotrophic factor(BDNF) and its specific tyrosine kinase receptor B(TrkB) are highly correlated to epilepsy. This study mean to explore the regulation of BDNF/TrkB signal pathway in hippocampal neurons by different isoforms of TrkB. Methods The cells were divided into 14 groups.The immunofluorescent technique was used to identificate the hippocampal neurons.Epileptiform discharges were detected by electrophysiological techniques.Western blot assay was used to determine the protein expression of TrkB and p-TrkB of each group. Results Compared with control group, the p-TrkB/TrkB value in control + BDNF group was higher. In epilepsy + BDNF group, the p-TrkB/TrkB value was lower than control+BDNF group, but higher than epilepsy group.The p-TrkB/TrkB value of epilepsy + ALLN + BDNF group was lower than epilepsy + BDNF group. The difference of the p-TrkB/TrkB value between epilepsy + ALLN + BDNF group and epilepsy + ALLN group was not significant.The p-TrkB/TrkB value in epilepsy + anisomycin + BDNF group was higher than both of epilepsy + anisomycin group and epilepsy + BDNF group. Conclusion BDNF can activate BDNF/TrkB signal pathway.The increased expression of TrkB.T can inhibit the activation of BDNF/TrkB signaling in status epilepticus.The change of TrkB.FL protein levels cannot inhibit the BDNF/TrkB signal pathway.