脑缺血区酸化调控神经干细胞分化的机制研究

Ischemic stroke, bearing the characteristics of high morbidity, lethality and mortality, has been becoming a significant problem in the medical domain. Thus, how to promote functional recovery of patients after ischemic stroke is the main challenge and focus to date. Endogenous neural stem cells (eNSCs) residued in the Subventricular zone (SVZ) could be in situ activated and proliferate, migrate to the lesion site, but only a few neural stem cells could differentiate into functional neurons might due to drastic change in local microenvironment. Hence, functional recovery is not satisfactory. Our previous study found that the lesion site always exist acidosis after ischemic stroke, and the eNSCs express acid-sensing ion channels 1a (ASIC1a) in SVZ. Meanwhile, acidosis could trigger intracellular Ca2+ elevation via ASIC1a homomeric channels thereafter promote eNSCs differentiation into astrocytes. These fingdings suggest that acidosis induced by ischemic stroke plays an important role in the differentiation of neural stem cells via activating ASIC1a channels. We will investigate the role and mechanisms of acidosis in the directional differentiation of neural stem cells through morphological methods, RNA interference and knock-out mice based on cultured eNSCs, brain slices and ischemic stroke animal model in the present project. And, the signaling pathway-Ca2+ elevation via ASIC1a homomeric channels to activate CaN-NLK-Wnt/β-catenin to modulate the differentiation of neural stem cells, is going to be systematically investigated. This study will not only help to elucidate the mechanisms controlling the directional differentiation of neural stem cells, but also provide evidence for the development of therapeutic strategy targeting it after ischemic stroke.

以高发病率及死残率为特点的缺血性脑卒中是医学领域的重大难题，如何促进脑缺血患者的神经功能康复是当前研究的难点和重点。室管膜下区的内源性神经干细胞可在脑缺血后发生原位增殖、向损伤灶处迁移，但因局部微环境剧烈改变致其在损伤灶处难以分化为足量功能神经元而阻碍神经功能康复。我们前期研究发现缺血区普遍存在微环境酸化，室管膜下区神经干细胞表达酸敏感离子通道1a，酸化可激活ASIC1a通道促发干细胞内钙升高，促进干细胞向胶质细胞分化。提示:缺血区酸化可激活干细胞上ASIC1a调控神经干细胞的分化。本项目拟通过形态学、RNAi、基因敲除等方法从细胞、脑片、动物模型等层次，系统研究缺血区酸化在神经干细胞分化调控中的作用，阐明“酸化-ASIC 1a-CaN-NLK-Wnt/β-catenin调控分化”这一机制。通过本项目的研究，不仅有助于深入阐明干细胞定向分化的调控机制，而且为设计新的干预靶点的奠定基础。

以高发病率及死残率为特点的缺血性脑卒中是医学领域的重大难题，如何促进脑缺血患者的神经功能康复是当前研究的难点和重点。室管膜下区的内源性神经干细胞可在脑缺血后发生原位增殖、向损伤灶处迁移，但因局部微环境剧烈改变可能致其在损伤灶处难以分化为足量功能神经元而阻碍神经功能康复。本研究基于前期研究成果，主要从微环境酸化对神经干细胞分化的影响、潜在机制及干预措施着手进行研究，发现：酸化可激活酸敏感离子通道1（ASIC1）开放，可促进神经干细胞分化为星形胶质细胞，给予ASIC1抑制剂Amiloride和/或PcTx1可有效逆转酸化促进神经干细胞分化为胶质细胞的效应。同时，使用分子生物学技术和基因Knockout技术发现抑制ASIC1表达也可有效逆转酸化促进神经干细胞分化为胶质细胞。同时，该课题研究了神经损伤后局部产物硫酸软骨素蛋白多糖（CSPG）对神经干细胞的影响及可能机制。此外，课题研究发现激活G蛋白偶联雌激素受体1（GPER1）可抑制神经干细胞增殖，但可促进神经干细胞向少突胶质细胞分化。另外 ，抗坏血酸（AA）或SVCT2过表达通过上调CDC42表达的方式促进F-actin组装，增强NSPCs迁移能力。通过该课题研究：发现了1个影响神经干细胞迁移和分化的损伤因素，2个可能的干预靶点或受体，3个可能用于脑缺血治疗的干预媒介，以期为脑卒中患者神经功能康复提供基础实验依据，为干细胞治疗脑卒中策略提供转化的可能性和理论学证据。