基于衰老的细胞模型研究PRDX1介导的信号通路在神经干细胞衰老中的调节作用

Aging refers to the physical and functional decline of the tissues over time which often leads to age-related degenerative diseases. Accumulating evidence implicates that the senescence of neural stem cells (NSCs) is of paramount importance to the aging of central neural system (CNS). However, exploration of the underlying molecular mechanisms has been hindered by the lack of proper aging models to allow the mechanistic examination within a reasonable time window. In the present study, we will optimize a hydroxyurea (HU) treatment protocol to induce sustained DNA damage without substantial apoptosis and aim to effectively induce postnatal subventricle NSCs to undergo cellular senescence. The aging of NSCs will be evaluated by senescence-associated-β-galactosidase (SA-β-gal) staining, their proliferation and differentiation capacity, the state of G0/G1 cell cycle arrest, the level of reactive oxygen species (ROS) level and cell death. We will also examine those classical cell-senescence genes as p16, p21 and p53 as well as the key protein involved in various DNA repair pathways such as xrcc2/3, ku70 et al. By this means, we are expecting to establish an in vitro NSC aging model and allow us to further perform quantitative proteomic analysis and outline the key regulators which may have profound impact on the aging of NSCs. Our preliminary data has implied the pivotal role of PRDX1 in NSC aging. In this study, we will further explore its molecular mechanism in depth by utilizing the conditional KO mouse of PRDX1 as well as our cellular aging model. Through these studies we will demonstrate the broad utilities of a cellular aging model and have a better understanding about the nature of NSC senescence and aging.

以往的研究显示神经干细胞的老化对神经系统的衰老起着重要作用。本研究拟通过优化羟基脲处理条件，诱导产生持续DNA损伤，通过SA-β-gal染色， 神经干细胞增殖分化能力，其在 G0/G1 细胞周期停滞状态，活性氧自由基的水平及细胞死亡的状况等指标评价神经干细胞的老化状态， 同时检测那些诱发衰老的关键基因(如p16,p21,p53)，以及DNA损伤修复及应答反应相关的重要蛋白xrcc2/3, ku70等的表达变化情况，从而确定是否成功地建立了神经干细胞衰老的离体细胞模型。在此基础上进行基于质谱的定量蛋白组学分析，筛选出调控神经干细胞衰老的关键因子。我们将以PRDX1为例， 利用条件敲除小鼠等分子遗传学手段，结合离体的衰老模型，首先确定PRDX1与神经干细胞衰老的关系，然后进一步研究PRDX1通过与p38 MAPK磷酸脂酶的相互作用对p38MAPK，p16的调控进而调节神经干细胞衰老的机制。

既往的研究显示神经干细胞的老化对神经系统的衰老起着重要作用，DNA损伤是细胞衰老的重要诱因，然而二者的相互关系在神经系统中乃至神经退行性疾病中仍待明确。在本项目中，我们首先建立了从年轻到年老的神经干细胞培养体系，体内研究发现小鼠神经干细胞库在衰老过程中减小，体外研究发现衰老小鼠的神经干细胞自我更新能力减弱、衰老程度增加，并且衰老小鼠的神经干细胞高表达炎症相关因子，通过神经干细胞衰老进程中蛋白表达的功能和通路分析以及关健分子网络与关健分子，我们发现神经干细胞在衰老进程中差异表达的基因主要参与神经系统疾病以及生物代谢过程相关。.在此基础上，我们通过DNA损伤诱导研究，发现HU处理诱导神经干细胞产生了广泛的DNA损伤、增加的细胞内活性氧水平以及降低的DNA损伤修复，我们的蛋白质组分析揭示急性DNA损伤主要造成神经干细胞蛋白质合成和线粒体功能的主要变化。基于神经干细胞衰老与神经系统疾病的相关性以及SIRT1参与细胞代谢调控的重要作用，我们进一步研究了帕金森疾病中SIRT1对神经干细胞早衰的影响，我们初步发现SIRT1通过自噬功能调节影响帕金森疾病中神经干细胞的早衰过程。.我们的研究为干细胞衰老与DNA损伤研究补充了新的知识，证明了DNA损伤能够通过细胞代谢影响神经干细胞的衰老进程以及神经退行性疾病，并揭示了神经退行性疾病如帕金森症中神经干细胞早衰的表型和初步的分子机制，为深入理解哺乳动物神经干细胞DNA 损伤修复机制以及为干细胞在预防神经性衰老疾病和再生医学中的应用奠定了新的理论基础。