星形胶质细胞上调基因-1（AEG-1）通过调控星形胶质细胞活化影响癫痫发病的作用机制研究

Astrogliosis is the important cause of epileptogenesis and repeat epileptic seizure. Study on the mechanism of the effect astrogliosis on epilepsy is one of the most heated topics. We previously proved that the astrocyte elevated gene-1(AEG-1) was over expressed in the hippocampuses of the epilepsy animal model, and that AEG-1 could induce astrogliosis. It is the first time for research in the mechanism of t effect of AEG-1 on astrogliosis by seizure. In the study, we assume that AEG-1 promote proliferation and migration of astrocyte to prevent the neuron regeneration by the pathway of TNF-α/AEG-1/NF-κB/vimentin, and on the other side, that AEG-1 destroy the glutamate uptake function to induce the death of astrocyte by the pathway of TNF-α/AEG-1/NF-κB/ EAAT2. To prove the hypothesis, our team carried out the following research: 1) Construct the induced AEG-1 knockout in astrocyte special mice model by using the CRISPR/Cas9 technology combining the Cre／LoxP recombination system. It provides a precise model for the subsequent study. 2) The effect of AEG-1 on the epileptogenesis and astrogliosis were investigated with the help of establishment of epilepsy animal model, EEG detection and immunofluorescence method. 3) The mechanism of the effect of AEG-1 on astrogliosis was studied by the method of astrocyte separation,Glutamate uptake assay and so on. In this way, we further extend the pathogenesis mechanism of epilepsy and give a new idea for development of anti-epileptic drug.

星形胶质细胞（AST）活化是癫痫发病的重要因素，探讨癫痫AST活化机制是目前研究重点。本项目前期证实AEG-1在癫痫大鼠海马中表达升高，且AEG-1过表达可诱导AST活化, 提示AEG-1在癫痫AST活化中的重要作用。本项目进一步提出AEG-1通过TNF-α/AEG-1/NF-κB/vimentin（EAAT2）途径促进AST活化增生和谷氨酸摄取功能受损，导致神经元再生障碍和死亡，进而引起癫痫发作。本项目拟开展研究如下：1）利用Cas9技术结合Cre／LoxP系统构建AST特异性AEG-1敲除小鼠，为后续试验提供精准模式动物；2）利用模式动物构建癫痫模型，采用脑电检测，免疫荧光等技术确证AEG-1在癫痫发生和AST活化中的作用；3）采用AST分离，谷氨酸摄取功能检测等方法探讨AEG-1相关具体分子途径在AST活化中的作用；阐明AEG-1在癫痫AST活化中的作用机制，为癫痫治疗提供潜在靶点。

目前癫痫的发病机制仍不清楚，抗癫痫药物不能从根本上治疗癫痫的发生，深入研究癫痫发生的分子机制显得日益重要。本项目组通过免疫荧光共染实验结果发现AEG-1在神经元和胶质细胞中皆有表达，且在神经元中的表达量明显高于在胶质细胞中的表达量，提示AEG-1在神经元中的功能不可忽视，因此本项目构建了海马皮层特异性敲除小鼠，在此基础上把AEG-1在颞叶癫痫中的作用机制研究从星形胶质细胞功能相关研究，扩展到了神经元和胶质细胞中的作用研究，从而更加全面地解析AEG-1在颞叶癫痫中的作用机制，进一步扩充癫痫发病机制，为癫痫药物治疗提供新思路。本项目取得的主要结果如下：1）成功建立了海马新皮层特异性敲除AEG-1小鼠；2）在此模型小鼠上，研究了AEG-1在颞叶癫痫发作中的作用及其机制，发现AEG-1的敲除可增强癫痫发作的易感性，这不仅与星形胶质细胞活化增强相关，且与小胶质细胞活化和海马CA1和CA3区神经元缺失相关；3）体外研究发现AEG-1过表达可诱导星形胶质细胞活化，并与TNF-α/NF-κB信号通路有关，AEG-1敲除可促进神经元凋亡，此结果提示海马皮层特异性敲除AEG-1后，癫痫易感性增加可能与神经元的功能失调有着更直接的联系。目前本项目发表中文核心3篇，培养两名研究生和两名本科生。本项目的核心研究结果正在撰写论文，以期2021年发表。项目负责人分别在2017年和2020年的宁夏医科大学国际医学高峰颅脑疾病分论坛中做相关的学术会议报告2次。