Activin A影响脊膜源性的GABA能神经祖细胞在体分化的机制研究

After the spinal cord injury, nerve regeneration is a major problem in the medical field. Glutamate excitotoxicity plays an important role in spinal cord injury, while enhanced the function of γ-aminobutyric acid (GABA) ergic neurons can effectively against excitotoxicity, and thus play a protective role. With the appearance of injury induced meninges-derived GABAergic progenitor cells and the increase of exogenous molecules Activin A after the spinal cord injury, it is possible for us to utilize of endogenous neurogenesis. However, these meninges-derived GABAergic progenitor cells originated in the nerve tissue or mesenchymal tissue has remained largely unknown, and it is not clear about the differentiation mechanisms of these meninges-derived GABAergic progenitor cells. In order to better understand the nerve regeneration, it is necessary to know the in vivo differentiation of the meninges-derived GABAergic progenitor cells. In this study, we will make all effort to clarify the in vivo differentiation of the meninges-derived GABAergic progenitor cells in cellular, organic and animal levels by using GAD67-GFP or GAD67-Cre transgenic mice, morphological, molecular biological, neurobiological and developmental methods. Meanwhile, the role of Activin A in the modulation of in vivo differentiation of meninges-derived GABAergic progenitor cells will also be investigated in this study. It is expected to offer more valuable information for the differentiation mechanisms of meninges-derived GABAergic progenitor cells and the establishment of therapeutic strategies for nerve regeneration after the spinal cord injury.

脊髓损伤的治疗是医学领域的重大难题，其根本原因在于中枢神经系统的再生困难。损伤诱导的脊膜源性的GABA能神经祖细胞的出现和外源性信号分子激活素A（Activin A）的增高，为利用内源性修复机制提供了可能。但是这些内源性神经祖细胞的来源和在体分化的调控问题尚待解决。本项目拟以脊膜源性的GABA能神经祖细胞为切入点，以GAD67-GFP和GAD67-Cre转基因小鼠为主要研究工具，借助逆转录病毒标记、神经形态学、发育神经生物学等方法，探索脊膜源性的GABA能神经祖细胞的组织起源，并进一步观察其在体分化特点。同时，综合利用分子生物学和行为药理学等方法，深入分析Activin A对脊膜源性的GABA能神经祖细胞在体分化的影响和对脊髓损伤修复的影响。我们期望在初步阐明Activin A参与脊膜源性的GABA能神经祖细胞在体分化的机制同时，为脊髓损伤后神经修复和功能恢复提供新的思路和策略。

由于中枢神经系统的再生困难，脊髓损伤的治疗一直是医学领域的重大难题。随着成体神经干细胞的发现和对神经干细胞增殖分化机制的深入研究，人们逐渐认识到神经干细胞可作为后备贮备，参与神经系统损伤修复。本项目旨在探讨损伤诱导的脑/脊膜来源的神经祖细胞的起源及其在体分化，为利用内源性修复和促进功能恢复提供新的思路和策略。取得的主要研究结果如下：① 脑/脊膜源性的神经祖细胞主要起源于脑室带。借助于多种转基因小鼠，免疫荧光双重标记结果表明在胚胎发育过程中，兴奋性（Tbr1、FoxP2、Brn1）和抑制性（PV、ENK）神经祖细胞标志物在脑/脊膜中一过性或持续性表达。进而胚胎电转结果表明在胚胎发育过程中，部分来自脑室带的增殖细胞可迁移至脑表面，提示这些脑膜源性的神经祖细胞可能起源自脑室带。② 脑/脊膜源性神经祖细胞具有可增殖性。借助杏仁核点燃模型和脊髓损伤模型，我们观察到神经损伤可诱导脑膜中神经祖细胞的增殖，同时脑/脊髓实质中亦出现部分增殖的神经元。③ 脑/脊膜源性神经祖细胞可在体分化为神经元。为了进一步特异性标记来自脑/脊膜源性的神经祖细胞，并进行在体追踪，借助质粒电转和tv-A/ASLV逆转录病毒标记系统，我们观察到脑/脊膜源性神经祖细胞可分化为典型的神经元。④ 借助GABA能神经元中条件性敲除mGluR5的小鼠，我们观察到mGluR5可能通过调控Activin A 受体（ACVR1）的表达参与脊髓损伤的修复。成年期脑/脊膜神经祖细胞的在体分化及其机制的揭示有助于增加人们对损伤修复时祖细胞来源的认识，有望为脊髓损伤后神经修复机制的阐明和关键分子的发现提供新的依据。