NDRG2通过Notch信号通路调控脑卒中后神经发生的分子机制研究

Cerebral ischemic stroke has been becoming the leading cause of mortality and disability in China, but until now, still lacks efficacious treatment. An accumulating evidence has shown that after cerebral ischemic stroke, endogenous neural stem cells (NSCs) in the subventricular zone can be mobilized to migrate into ischemic region, involving in neuronal repair. Therefore, how to effectively enhance the proliferation, differentiation and survival of NSCs, ultimately promoting the repair of neurological functions, are the urgent issues for us to address. In our recent studies, we demonstrated that NDRG2, a kind of tumor-suppressing factor, was expressed in the NSCs in the neurogenic zones of adult rats. Importantly, its expression was upregulated after cerebral ischemic stroke. Manipulation of NDRG2 expression could affect the expression of downstream effectors of Notch signaling, a crucial pathway for the regulation of NSC neurogenesis, strongly implying that NDRG2 may act on post-stroke neurogenesis by regulating Notch signaling. To further investigate the role of NDRG2 in the neurogenesis after stroke, in this project we will take advantage of viral transfection techniques to manipulate NDRG2 expression in ischemic stroke models in vivo or in vitro, then observe the changes in proliferation, differentiation and survival of NSCs, and examine the expression of Notch signaling-associated molecules as well; Using co-immunoprecipitation and pharmacologic intervention, we will explore the regulatory relationships between NDRG2 and Notch; Using ndrg2 knockout mice, we will investigate whether ndrg2 deletion can affect animal’s neurological functions. Our results will uncover a key role of NDRG2 in adult neurogenesis after ischemic stroke, and will also provide the original experimental basis for clinical application of NSCs in the treatment of cerebral ischemic stroke.

脑卒中已成为我国第一大致死和致残病因，然而至今尚缺乏有效治疗手段。研究表明，脑卒中后机体可动员内源性神经干细胞（NSCs）迁移到缺血损伤区，参与损伤的修复。然而如何有效促进NSCs增殖、分化及存活，最终促进神经功能恢复是亟待解决的关键问题。我们近期发现，一种肿瘤抑制分子NDRG2表达于脑内神经发生区的NSCs，缺血后其表达显著升高；干预NDRG2可影响神经发生关键调控分子Notch的表达，强烈提示NDRG2可能通过调控Notch信号参与缺血后的神经发生。为此，本课题拟在体内、体外缺血和缺氧模型上，通过病毒感染等手段改变NDRG2表达，观察NSCs增殖、分化、存活及Notch表达的变化；利用免疫共沉淀等技术揭示NDRG2和Notch之间的作用及调控关系；利用基因敲除小鼠观察ndrg2缺失对脑缺血后神经功能的影响，以揭示NDRG2对神经发生的关键作用并为临床应用NSCs治疗脑卒中提供实验依据。

脑卒中已成为我国第一大致死和致残病因，然而至今缺乏有效治疗手段。研究表明，脑卒中后内源性NSCs可迁移到缺血区，参与损伤的修复。因此探寻有效调控NSCs命运的关键分子，可能成为今后治疗脑卒中最具前景的策略之一。本项目旨在探讨表达于脑内NSCs的一种抑癌分子—NDRG2，在脑缺血损伤时对神经发生的作用及调控机制。在国家自然科学基金（No. 81571223）的资助下，借助大鼠MCAO缺血模型和体外OGD模型，通过病毒感染等手段改变NDRG2表达，观察NSCs增殖、分化、存活及Notch表达的变化，并探索NDRG2和Notch之间的作用及调控关系；利用ndrg2-/-小鼠观察ndrg2缺失对脑缺血后神经发生的影响。通过上述研究，获得了以下主要结果：（1）利用免疫荧光染色技术，发现NDRG2表达于SVZ神经发生区NSCs（Nestin+）并主要在分裂期的NSCs（BrdU+和Ki67+）中；进一步发现NDRG2表达在GFAP+ B型和Sox2+ C型NSCs细胞但不在DCX+ A型细胞中；（2）利用免疫印迹和免疫组织化学染色，发现脑缺血后NDRG2在SVZ区的表达显著升高，与Nestin+和Sox2+细胞数目同步增加；（3）利用Real-time PCR技术，对Notch信号通路各个分子，包括Notch配体（Dll1、Dll4、Jagged1和Jagged2）、Notch受体（Notch1~4）、Notch受体胞内段NICD、RBPJ、靶分子Hes1和Hes5进行了检测，发现在SVZ区Notch1和Jagged2的表达水平较高；（4）利用免疫荧光三重染色，发现NDRG2表达在Jagged2+但不在Notch1+细胞中，NDRG2+/Jagged2+双标记细胞与Notch1+细胞在空间上紧密相临；抑制Notch可减少NSCs数目；（5）通过病毒感染调控基因表达和免疫印迹技术，发现在体和离体过表达ndrg2可激活Notch信号通路，表现为Notch靶分子Hes1表达增高，同时可促进NSCs的增殖；沉默ndrg2表达则抑制Notch信号并减少NSCs增殖；（6）利用免疫组织化学染色，发现ndrg2-/-小鼠SVZ区增殖细胞数目明显少于野生型小鼠。以上结果初步揭示了NDRG2可能通过调控Notch信号参与动物脑缺血损伤后的神经发生，为临床应用NSCs治疗脑卒中提供了实验依据。