SIRT1对小鼠神经干细胞DNA损伤修复的作用机制

Neural stem cells (NSC) aging is closely related to senile nervous system diseases. The decrease of DNA damage repair capacity is an important incentive to induce stem cell aging. Yet At present, the mechanisms about the NSC DNA repair defect prompting aging is unclear. Our preliminary work found SIRT1 upregulated in the aging model induced by DNA damage. Meanwhile, DNA damage increased after inhibiting the expression of SIRT1. Then the speculation was obtained by both results that SIRT1 participated in NSC DNA damage and repair process. Based on the preliminary data, this project will detect the role of SIRT1 on the NSC DNA damage and repair by interfering SIRT1 expression in vitro and in vivo conditional knockout SIRT1. For the mechanism research, firstly, no-homologous end joining, NHEJ efficiency of SIRT1 will be detected by DNA double-strand break (DSB) repair reporter system. Secondly, the relationship between SIRT1 deacetylation KU70 and NHEJ will be researched by co-immunoprecipitation. Lastly, the effect of SIRT1 deacetylation KU70 on KU downstream elements combining to DNA ends will also be uncovered. The purpose of this project is to explain the role of SIRT1 on DNA damage repair and related regulatory mechanisms in mice NSC. This will lay the theoretical foundation for the applied study of NSC regeneration and anti-aging.

神经干细胞（NSC）衰老与老年神经系统疾病密切相关，DNA损伤修复能力降低是干细胞衰老的重要诱因，而目前有关NSC DNA损伤修复缺陷促使衰老的机制尚不明。前期工作发现，在DNA损伤诱导形成的小鼠NSC衰老模型中，SIRT1表达上调；抑制SIRT1表达，NSC DNA损伤程度增加，由此推测SIRT1可能参与NSC DNA损伤修复过程。本项目拟通过体外干扰SIRT1表达、体内条件性敲除SIRT1的方法，检测SIRT1对NSC DNA损伤进行修复的作用；利用DNA双链断裂（DSB）修复报告系统检测SIRT1参与非同源末端连接（NHEJ）的效率；利用免疫共沉淀的方法检测SIRT1去乙酰化KU70参与NHEJ修复及其对KU70下游元件结合DNA损伤末端的调控作用。旨在阐释SIRT1对小鼠NSC DNA损伤修复的作用及调控机制，为NSC的再生及抗衰老的应用研究奠定理论基础。

神经干细胞（NSC）衰老与老年神经系统疾病密切相关，DNA损伤修复能力降低是干细胞衰老的重要诱因，而目前有关NSC DNA损伤修复缺陷促使衰老的机制尚不明。我们分别在细胞衰老模型、自然衰老状态下检测了小鼠SVZ区NSC的基因组稳定性及衰老的表型，并通过蛋白组测序得到多个可能参与调控基因组稳定性的基因，其中包括SIRT1基因。SIRT1是一种NAD+依赖的去乙酰化酶，能够通过染色质修饰、沉默基因的表达以及对DNA损伤进行修复等多种方式维持基因组稳定性。在NSC衰老过程中,SIRT1表达上调；课题通过体内、体外多个水平验证了SIRT1能够通过维持基因组稳定性调控NSC细胞衰老的作用。为寻找SIRT1调控NSC细胞衰老的作用机制，我们利用SIRT1抗体进行了co-IP-MS实验，并发现多个与SIRT1相互作用的蛋白。后续实验将对这些蛋白进行验证，并确定SIRT1调控基因维护基因组稳定性，从而延缓衰老的作用机制。