灵芝孢子有效成分筛选及与苔藓纤维发芽相关蛋白构效关系研究

The mossy fiber sprouting plays an essential role during the genesis and progression of epilepsy. It is clearly that several cellular molecules interrupt this motion through the Trk signal transduction pathway, but the detailed molecular mechanisms are still not clear. As our previous work, the lucidum spores are determined to be responsible for the inhibitory activity to the mossy fiber sprouting in epileptogenesis. In order to further elucidate the mechanisms and bioactivity of constituents, in this work, the effective components of lucidum spores will be selected by the in vitro and in vivo anti-epilepsy activity experiments. On such basis, with the aid of the Western blot and laser scanning confocal microscope methods, the changes of cellular molecules within the Trk signal transduction pathway will be explored to clarify the corresponding inhibiting mechanisms. Further, the electrophysiological activity will be detected by the patch clamp recording technique. The effects involving that lucidum spores inhibited the mossy fiber sprouting on the preventing of epileptogenesis will be validated at the levels of animal and cell, associated with the analysis of bioactivity constituents. We anticipate that the results will aid understanding the pathogenesis of epileptic mossy fiber channel, finding the novel anti-epilepsy targets, fully utilizing the lucidum spores, as well as preventing and treating the epilepsy.

癫痫后海马苔藓纤维出芽(MFS)和突触重组是癫痫发病机制之一。本课题组首次发现灵芝孢子具有抗癫痫作用，并证实它抑制MFS，但是，灵芝孢子抑制MFS的机制，迄今国内外尚无报道。在前期研究中通过结构域功能分析、同源序列多重比对、Go分类等手段，发现灵芝酸是微管蛋白的有效抑制剂。本研究拟证实灵芝孢子天然小分子活性物质是否通过抑制MFS具有抗癫痫的效应，采用"组分筛分天然活性物质发现体系"筛选灵芝孢子天然小分子活性物质，通过激光共聚焦显微镜、流式细胞仪、生物芯片、Western blot等技术寻找靶蛋白；运用分子对接和分子动力学方法研究灵芝孢子单体与蛋白的相互作用，并期望阐明灵芝孢子中何种单体成分具有抗癫痫的作用，构建出以细胞分（因）子-Trk信号转导途径-苔藓纤维发芽-癫痫为核心的灵芝孢子抗癫痫作用分子网络，阐明灵芝孢子抗癫痫分子机制；为寻找理想的抗癫痫药物提供新的靶点。

癫痫后海马苔藓纤维出芽(MFS)和突触重组是癫痫发病机制之一。本课题组首次发现灵芝孢子具有抗癫痫作用，并证实它抑制MFS，但是，灵芝孢子抑制MFS的机制，迄今国内外尚无报道。在前期研究中通过结构域功能分析、同源序列多重比对、Go分类等手段，发现灵芝酸是微管蛋白的有效抑制剂。本研究通过激光共聚焦显微镜、流式细胞仪、Western blot等技术寻找靶蛋白；运用分子对接和分子动力学方法研究灵芝孢子单体与蛋白的相互作用，实验证实灵芝多糖能增强大鼠海马神经元Cav-1表达和抑制NF-κB的表达；并通过提高BDNF和Survivin的表达进而起到保护神经元的作用；甜菜碱可以增加γ-氨基丁酸受体的表达具有抗癫痫作用；灵芝酸DM可以修复海马神经元细胞异常放电引起的损伤，抑制海马神经元异常放电的作用与其稳定微管蛋白的作用有关。本研究为寻找理想的抗癫痫药物提供新的靶点。.迄今为止，在国内外已发表标记本课题资助的研究论文6篇；获实用新型专利2项；待发表论文1篇。培养硕士研究生5名。