造血干细胞衰老的细胞记忆机制研究及熟地甘露三糖的干预作用

It has been proved that hematopoietic stem cells (HSC) aging is closely related to aging and geriatric diseases, bone marrow hematopoietic suppression and even leukemia, so anti-aging of HSC would have major significance to explore the aging mechanism and prevent and treat of related diseases. At present, the research about mechanism of hematopoietic stem cell aging rarely related to cellular memory —— when the external environment of stem cells changes briefly, stem cells will remember the stimulate, through gene expression regulation, permanent differentiates the particular organization cells. It has been reported oligosaccharide in Rehmannia which is the “the prince of hemopoietics” can promote the HSC proliferation and differentiation, based on our previous experimental results such as mannotriose, as the Quality control indicators of Rehmannia, could resist the aging of hippocampal neuron , this topic will explore HSC aging mechanism from cellular memory angle and study the influence of manninotriose to HSC aging. In the topic, establlish aging HSC model in vitro, stem cell aging related conventional indicator, cellular gene expression of the key cell memory protein in transcription inhibitors PCG and transcription activator TrxG were detect ed to observe the changes in aging HSCs and the effect of mannotriose on the oboved indicator. We also use RNAi technology to make clear the key gene. The study aimed at developing new drugs which can resisit stem cells aging and its possible biological mechanism, and then providing a experimental basis for anti-aging and prevention and treatment of geriatric diseases.

已证明造血干细胞（HSC）衰老与机体衰老及老年病、骨髓造血抑制甚至白血病有密切联系。故延缓HSC衰老对阐述衰老机制及防治相关疾病有重大意义。目前造血干细胞衰老的机制研究很少涉及细胞记忆角度—即当干细胞外环境短暂改变时，干细胞会记忆刺激，通过基因调控，永久性分化为特定成熟组织细胞。有报道“补血之君”熟地的寡糖能促进HSC增殖分化，结合我们前期的熟地寡糖中质量控制指标甘露三糖具有抗细胞衰老作用等相关结果，本课题将对HSC衰老的细胞记忆机制做以初探并研究甘露三糖的抗HSC衰老作用。本课题拟建立HSC衰老模型，研究甘露三糖对HSC衰老相关指标及细胞记忆蛋白中转录抑制因子PCG家族和转录激活因子TrxG家族的关键基因及靶基因表达的影响，并通过RNA干扰技术明确HSC衰老的关键基因。本课题旨在寻找能激活衰老干细胞的药物及其可能的生物学调控机制，从而为延缓衰老和防治老年病提供新的实验支持。

中文摘要.目的：探讨HSC衰老的细胞记忆机制，从而为丰富衰老的机制学说及寻找抗HSC衰老的药物提供实验依据。方法：MACS体外分离HSC/HSPC，流式细胞术检测细胞纯度。用含有10ng/ml IL3+10ng/ml IL6+30ng/ml SCF的干细胞培养基培养HSC/HSPC 6-8天建HSPC衰老模型。通过以下指标鉴定模型：SA-Gal染色，细胞周期，CFU-Mix，蛋白组学（ITRAQ）分析。进一步观察黄芪多糖,甘露三糖对衰老HSPC相关指标及细胞凋亡的影响；Rt-PCR、WB检测衰老HSC/HSPC中细胞记忆相关PCG 家族和TrxG 家族的主要蛋白，H3K27me3和H3K4me3基因表达变化；siRNA干扰技术敲降细胞记忆蛋白Rae-28和TRX,研究其是否影响HSC的衰老。结果： Lin-c-kit+细胞即HSPC具有较高的纯度；与年轻组相比，衰老组细胞：SA-Gal阳性细胞百分比，G0/G1的细胞比例明显升高；多向分化能力显著降低； aging代谢，糖酵解，溶酶体代谢，核糖体合成及mRNA剪接显著变化；以上结果说明模型为衰老的HSPC。黄芪多糖显著逆转上述衰老指标，且抑制细胞凋亡。甘露三糖组HSPC中：SA-β-半乳糖苷酶阳性细胞百分比及凋亡细胞的比例并无显著性差异；混合集落形成能力显著提高；cd117+sca-1+细胞显著增高，提示甘露三糖可以提高衰老过程中HSC的比例。细胞记忆相关蛋白及H3K27me3，H3K4me3表达均显著降低。.结论：本研究获得了一种新的快速致HSPC衰老的体外培养平台。黄芪多糖可对抗HSPC的凋亡和衰老。甘露三糖在HSC衰老过程中具有潜在的增强HSC干性的作用。衰老HSPC中细胞记忆相关蛋白PCG和TrxG家族的基因表达发生了改变，通过下调H3K27me3，H3K4me3水平调 控靶基因，破坏了防止细胞身份发生改变的细胞记忆系统平衡从而致衰老。