Kv1.3离子通道功能变化调节神经干细胞成熟与分化的机制研究

Potassium ion channels participate multiple biologic activities. Kv1.3 potassium channel is widely expressed on neural system, especially on neural stem cells (NSCs). NSCs are an important and unlimited donor source for cell replacement therapy in the treatment of many neurological diseases, but its differentiation efficiency is low and the mechanism is unclear. Our previous research study revealed that the efficiency of neural differentiation from neural stem cells was enhanced by Vitamin E isomer ∂-tocopherol via Ca2+ channels. Moreover, we found that Kv1.3 blockers induced a significant increase in the proportion of GABA-positive cells. Therefore, we hypothesis that Kv1.3 blockade enhances presynaptic neurotransmitter release, induces postsynaptic membrane depolarization and GABA receptor activation, and then increases excitatory postsynaptic currents (EPSCs) efficiency and intracellular Ca2+ concentration， which can promotes neuronal differentiation. In this project, we explore the effects of Kv1.3 channel on NSC neuronal differentiation and the functional relationship between Kv1.3 and GABA signaling using molecular and cell biology、immunocytochemistry and electrophysiology approaches. It will be beneficial for understanding the mechanism of neural stem cell differentiation, and may provide a new method to obtain more mature and functional neurons from neural stem cells for clinical application.

钾离子通道参与多种生命活动，Kv1.3在神经系统，特别在神经干细胞上分布广泛。神经干细胞兼有神经修复和再生功能，有望用于治疗神经损伤和神经退行性疾病，但干细胞定向分化效率较低，其分化机理不明。我们前期研究发现维生素E能调节钙离子通道，促进神经干细胞分化；我们进一步发现其分化过程中加入Kv1.3阻断剂，能获得更多更成熟的GABA阳性神经元，由此我们推测:阻断Kv1.3引起突触前神经递质GABA释放增多,使突触后神经前体细胞钙离子内流增加, 从而促进其向神经元方向分化。本项目在前期实验基础上,以研究离子通道Kv1.3功能与GABA信号的关系为主线,构建神经干细胞体外分化体系,利用分子生物学、膜片钳技术和RNA干扰等实验手段,探索Kv1.3与GABA信号系统在神经干细胞分化过程中的功能联系。本项目的研究为深入理解神经干细胞的分化机理提供新的理论,为获取更多的成熟神经元及其临床应用提供新的方法。

本项目采用小鼠神经干细胞为研究对象，研究Kv1.3离子通道对神经干细胞定向分化的影响，通过shRNA基因沉默技术对Kv1.3的表达进行干扰，与正常的神经干细胞进行形态和功能的对比，分析神经干细胞的分化情况。其次，通过加入Kv1.3离子通道选择性阻断剂，与正常分化的神经干细胞进行对比，分析神经干细胞的活性及分化情况。在实验过程中合成小分子化合物Kv1.3选择性阻断剂Psora-4，并用Psora-4抑制KV1.3离子通道进行神经干细胞分化成熟的研究。通过实验证明选择性阻断和沉默神经干细胞中的KV1.3离子通道能够促进其向神经元方向分化，Psora-4能够从形态和功能两个方面显著促进新生神经元的成熟。通过研究发现Kv1.3与GABA信号系统在分化过程中跟GABA的功能有关系，下一步再通过电生理实验证明其内在的联系。