利用慢病毒-shRNA文库体内筛选技术研究限食延缓造血干细胞衰老的靶点和机制

Given that hematopoietic stem cells (HSCs) play essential roles in maintenance and regeneration of the hematopoietic system, it is believed that HSC aging has major impact on hematopoietic system aging. Dietary restriction (DR) is the most prominent non-pharmacological anti-aging intervention. Previous work of the applicant in Germany has provided the first experimental evidence that DR ameliorated HSC aging phenotypes such as the increase in number of HSCs and the skewing towards myeloid-biased HSCs during aging (J Exp Med., 2016). However, the underlying molecular mechanism remains to be delineated. It has been shown that accumulation of damage due to oxidative stress in cells is an important contributor promoting aging. Furthermore, our preliminary experiments indicate that DR significantly reduces reactive oxygen species (ROS) in HSCs. Taken together we hypothesize that DR retards HSC aging through reduction of ROS. To address the hypothesis, the project intends to study the effect of DR on ROS regulation and its role in HSC aging using oxidizing and anti-oxidizing agent. Furthermore, the study will establish lentivirus-shRNA library targeting oxidative stress related genes, and seek to identify novel targets of DR that significantly impact on HSC aging by in vivo functional screening. The identified candidates will be further verified for its role on HSC aging. In summary, the project will deepen the understanding of the molecular mechanism of how DR ameliorates HSC aging, therefore provide theoretical basis for proper application of DR to improve overall population health in the “aging” society.

造血干细胞衰老是造血系统衰老的最重要成因，限食是最肯定的非药物延缓衰老的手段。申请人在德国期间的研究表明：限食能全面延缓造血干细胞衰老的表型和功能（J Exp Med. 2016），但相关调控机制尚待阐明。我们的预实验提示限食显著降低造血干细胞的氧化应激水平,而氧化应激是导致衰老的重要机制之一。据此我们假设：限食通过改善氧化应激以延缓造血干细胞衰老。为验证该假说及深入探索限食调控氧化应激的靶点，本课题拟在前期工作的基础上，通过应用氧化剂与抗氧化剂研究氧化应激在限食延缓造血干细胞衰老中的作用；建立氧化应激相关基因慢病毒-shRNA文库，体内功能性筛选限食改善造血干细胞氧化应激及延缓其衰老的关键靶点；进一步分析及验证筛选出的靶基因对造血干细胞衰老的作用，从而探索改善氧化应激以干预造血干细胞衰老的新方法。本课题将为“老龄化”社会形势下合理应用限食、提高人群健康指数提供理论指导。

目前，世界老年人口正在迅速扩增，我国与许多发达国家一样面临人口老龄化的严峻挑战。多种造血系统疾病在老年人中发病率显著提高。不仅如此，在对许多血液病有根治希望的造血细胞移植中，供者年龄与移植预后呈负性相关。上述问题主要与衰老造血干细胞（HSC）异常有关。我们曾经报道始于青年的限食可显著延缓HSC衰老，但限食是否能改善甚至逆转HSC衰老尚无报道。本项目通过流式细胞染色、小鼠造血干细胞/骨髓移植、实时定量PCR等方式，发现起始于中老年的限食可明显逆转HSC衰老的表型，短期限食可改善老年供者来源的HSC移植后重建，促进重建后造血系统淋、髓系平衡，但对未经分选的造血细胞移植未体现出改善作用。长期限食可进一步增强年轻化HSC的作用，能明显改善老年小鼠供者来源的造血细胞移植。其分子机制与限食上调衰老HSC中SIRT2、SIRT7，减少mtUPR相关。这项研究成果不仅为HSC衰老的机制提供了重要新知，而且对临床实践中，供者年龄偏大情况下的造血细胞移植有重要的启示意义，将会具有重要的临床转化前景。.DNA损伤积累是干细胞衰老的一大机制。放射线照射可引起DNA损伤，使HSC产生衰老相关表型，因而可作为研究HSC衰老的一种模型之一。本项目在我们建立的慢病毒-shRNA文库中包含了Wnt信号途径相关基因，并在放射损伤模型中筛选出了LRP6这个可能对DNA损伤中HSC命运起关键作用的基因。我们发现，照射后HSC中Wnt活性水平先升高后降低，与DNA损伤信号途径一致。我们进一步通过体外和体内研究表明，Wnt活性对DNA损伤的HSC命运起决定作用，通过敲降LRP6降低Wnt信号途径活性能更好的保护DNA损伤的HSC。这些研究成果对理解衰老过程中HSC累积DNA损伤后引起的分子改变提供了重要线索，为干细胞衰老及相关的肿瘤形成机制提供了新的认识，对探索延缓HSC衰老、相关肿瘤防治等有重要提示意义。