下调p38MAPKα-Mitf-嘌呤与mTORC1-SREBP1-脂质合成轴阻止扩增人HSCs的衰老以维持干性

The widely application of hematopoietic stem cells (HSCs) is restricted due to the insufficient cell numbers. Although HSCs could be obtained via ex vivo expansion,their stemness fails to be maintained by current expansion regimens.Our previous projects (NSFC 30871097# and 81170524#) have confirmed that：①the senescence is one of the mechanisms of the stemness damage in expanded HSCs;②the activation of both p38 mitogen activated protein kinase α(P38MAPKα) and complex 1 of the mammalian target of rapamycin (mTORC1) is still observed in StemRegenin 1 (SR1,a differentiation inhibitor) expanded HSCs,and the down-regulation of both P38MAPKα and mTORC1 could inhibit senescence and improve short-term hematopoietic reconstitution capability of the expanded HSCs;③the combined down-regulation of P38MAPKα and mTORC1 also decreases the expression of microphthalmia-associated transcription factor (Mitf) and sterol regulatory element binding protein 1 (SREBP1) in ex vivo expanded HSCs.Current studies have proved that cellular senescence could be induced through up-regulating purine and lipid synthesis performed by increased expression of Mitf and SREBP1 respectively. Therefore, the following questions will be investigated in this project:①whether the long term hematopoietic reconstitution ability of SR1 expanded HSCs could be maintained through down-regulation of both P38MAPKα and mTORC1 activities;②whether the senescence of expanded HSCs is prevented through the down-regulation of both p38MAPKα-Mitf-purine and mTORC1-SREBP1-lipid synthesis axes.

造血干细胞（HSCs）数量不足限制其广泛应用，体外扩增有望解决此问题，但现有方案难以维持其干性。我们以往研究(NSFC 30871097# ，81170524#)证明：①扩增HSCs衰老是其干性丧失的机制之一；②分化抑制剂（SR1）扩增的HSCs存在p38丝裂原活化蛋白激酶α（P38MAPKα）和哺乳动物雷帕霉素靶蛋白（mTORC1）的活化，两者的联合下调可抑制其衰老并改善短期造血重建；③两者的联合下调也可降低扩增HSCs的小眼相关转录因子（Mitf）和胆固醇调节元件结合蛋白（SREBP1）水平。文献证实：高水平的Mitf和SREBP1可分别通过上调嘌呤和脂质合成诱导衰老，为此本项目拟研究：联合下调活化的P38MAPKα和mTORC1能否维持SR1扩增的HSCs长期造血重建能力，是否通过p38MAPKα-Mitf-嘌呤与mTORC1-SREBP1-脂质合成轴的下调阻止扩增HSCs的衰老。

目前的方法通常不能维持体外扩增人HSCs的干性，为了探索分化抑制剂Stem Regenin 1 (SR1， 芳香烃受体拮抗剂) 扩增的人脐带血CD34+ 细胞源HSCs的干性维持失败机制，我们调查了SR1扩增的人脐带血CD34+ 细胞的p38丝裂原活化的蛋白激酶α(p38 MAPKα, p38α)和哺乳动物雷帕霉素复合物1靶蛋白 (mTORC1) 的活性与影响;结果显示：SR1扩增的人脐带血CD34+ 细胞发生衰老与p38 MAPKα和mTORC1的先后活化；它们的共同抑制导致扩增的人脐带血CD34+ 细胞衰老减少而不影响凋亡，促进了扩增表型HSCs的维持但未抑制分化，促进了它们在NOG（ NOD/Shi-scid/IL-2Rγnull ）小鼠的多系造血重建与长期自我更新能力。我们的机制研究揭示：我们的扩增体系诱导的衰老抑制主要归因于剪接体、蛋白酶体形成与嘧啶代谢信号通路的下调。这些结果提示：活化的p38 MAPKα和mTORC1的共同下调通过衰老抑制增强了SR1扩增人脐带血CD34+ 细胞源HSCs的干性维持。因此，我们建立了一个新的策略--通过同时抑制多个独立的衰老始动信号以维持体外分化抑制剂扩增的人HSCs的干性，这种衰老抑制诱导的体外扩增HSCs的干性维持可能在其它的HSCs扩增系统也有重要的作用。