DNA双链断裂修复基因Nbs1在造血干细胞维持和衰老中的功能研究

Tissue homeostasis in adult animals is maintained by the tissue specific adult stem cells. They self-renew to sustain stem cell pool and differentiate to generate functionally committed progenies to maintain tissue integrity. DNA damage is one of the key factors in quality control of adult stem cells. In mammals, DNA damage responses are the key mechanisms to maintain the genomic stability and thus to prevent the organismal ageing. DNA double strand break (DSB) repair gene Nbs1 senses the DSB, and activates the DSB repair signaling pathway. Mutation of NBS1 in human causes autosomal recessive disorder Nijmegen breakage syndrome, characterized by growth retardation, microcephaly, immunodeficiency, lymphoma predisposition and a shorter life span. Previously we have generated mouse models with central nervous system and T cell specific depletion of Nbs1, Mechanistically，Nbs1 is essential to repair those physiologically generated DSBs and thus safeguards the proper development of brain and thymus. However, a direct link of Nbs1 in adult stem cell maintenance and ageing process is still missing. In this application, by generating the mouse model with Nbs1 specific deletion in hematopoietic stem cells (HSCs), we propose to functionally characterize the role of Nbs1 mediated DSB repair pathway in HSC maintenance and ageing. Furthermore, we intend to investigate cell fate determination mechanisms on HSCs and their progenies responding to Nbs1 null generated DSBs. Our study could, not only, fill in the knowledge gap of Nbs1 in HSC maintenance, but also shed light on the contribution of DNA damage response to HSC ageing. In addition, this research may be useful for the mechanistic control in ex vivo expansion of HSCs for therapeutic applications.

成体干细胞是维持生物体组织动态平衡，防止机体衰老和老年退行性疾病的重要因素。成体干细胞中DNA损伤的累积破坏其自我更新和分化能力，促进生物体衰老。DNA损伤应答反应是维护细胞基因组稳定性的核心机制。Nbs1识别和修复DNA双链断裂。人类NBS1突变导致Nijmegen 断裂综合症，其临床症状是小头畸形，免疫缺陷、多发性淋巴瘤，且病人多在青壮年早衰死亡。前期研究揭示Nbs1通过修复生理状态下产生的DSB来确保神经系统和胸腺正常发育。但是Nbs1在衰老中机制尚不明确。造血干细胞(HSC)稳态维持是防止机体衰老的保障。本项目拟采用HSC特异性Nbs1敲除小鼠，研究Nbs1介导的DNA损伤修复机制在HSC稳态维持和衰老中重要作用，揭示HSC面对DNA损伤信号时细胞命运抉择及其分子机理。研究成果将填补Nbs1在HSC研究中的空白，加深对DNA损伤与HSC衰老的认识，同时为HSC体外扩增提供新思路。

组织特异性干细胞，通过其自我更新和分化能力，维持组织稳态和生命有机体年轻态。基因组稳定性维持是保证组织特异性干细胞自我更新和分化以及对抗衰老的重要机制。DNA双链断裂修复机制是维持细胞基因组稳定性的重要保障。本研究以DNA双链断裂修复基因Nbs1为切入点，以造血干细胞（HSC）为研究对象，采用两种方式构建了Nbs1在HSC中特异性敲除小鼠，研究了DNA双链断裂修复机制在HSC自我更新/分化，以及造血系统稳态维持和衰老中功能。研究发现：.1.构建了Nbs1在成体HSC中诱导型敲除小鼠模型（Nbs1F/FMx-Cre+）。研究揭示，在成年小鼠中诱导Nbs1敲除造成骨髓中造血细胞数目锐减，造血前体细胞比率降低，但LSK细胞比率无变化，长程HSC（CD34-LSK，LT-HSC）比率略有增加。同时，Nbs1敲除造成本应处于静息状态的LT-HSC中DNA损伤累积，进入细胞周期进行增殖。.2.构建了Nbs1在胚胎造血干细胞中特异性敲除小鼠（Nbs1F/FVav-Cre+）。研究发现Nbs1在胚胎HSC中敲除导致约50%的突变小鼠在胚胎期死亡，可出生的突变小鼠在出生11天内由于造血系统衰竭而死。Nbs1敲除造成胚胎肝脏中造血系统发育异常，成熟造血细胞/造血前体细胞数目急剧降低，但是HSC数目存在显著升高。对HSC深入分析表明：1）相对于野生型小鼠中活跃增殖的HSC，Nbs1缺陷型HSC过早退出细胞周期，这可能是由于Nbs1缺陷型HSC中存在低度的p21升高，导致细胞周期抑制所致；2）HSC和造血前体细胞应对DNA双链断裂存在不同的命运决定机制：HSC倾向于激活细胞周期阻滞从而起到自保；而造血前体细胞通过表达高程度的细胞凋亡因子起始细胞死亡命运；3）Nbs1敲除造成造血系统中DNA损伤修复反应（如p53通路）激活，但是p53敲除却不能拯救Nbs1缺陷型小鼠的死亡，反而加速其死亡。.综上，DNA双链断裂修复机制是HSC增殖和稳态维持的重要调控机制，其功能缺失导致造血系统稳态失衡和小鼠生命力降低；结合已发表人类脐血HSC和小鼠成体HSC的研究数据，我们的研究表明人和小鼠胚胎来源HSC和造血前体细胞在应对DNA损伤时存在命运差异，人脐血HSC面对DNA损伤时倾向于细胞凋亡；而小鼠HSC，不论成体和胚胎来源，均趋向于细胞周期阻滞。