SphK2调控造血干细胞自我更新和损伤修复机制的研究

To promote the self-renewal and hematopoietic reconstitution of hematopoietic stem cells (HSCs) after injury and bone marrow transplantation need to be addressed urgently, and was restricted by the research on the self-renewal and post-injury regeneration mechanism of HSC. The applicant found SphK2 played a crucial role in hematopoiesis and injury recovery via genome-scale CRISPR-Cas9 knockout screening assay. SphK2 knockout could promote HSC self-renewal significantly under both normal and chemotherapy-induced conditions. SphK2 knockout mice with chemotherapy showed accelerated HSC self-renewal, reduced mortality and increased hematopoietic reconstruction. These findings indicate that SphK2 is crucial to HSC self-renewal and injury recovery. Previous studies showed that nuclear SphK2 could be stimulated to catalyze Sphingosine-1-Phosphate (S1P) to repress histone acetylation. We will investigate transcriptome and epigenome of HSCs based on established methods of rare cell populations to explore the mechanism of HSC self-renewal via SphK2, in order to provide insight into HSC therapy and potential molecular targets for clinical applications.

如何提高造血干细胞（HSC）的自我更新能力促进造血系统放化疗损伤后修复和骨髓移植后造血重建是目前临床亟待解决的问题。因此，研究HSC化疗损伤后自我更新能力调控的分子机制具有重要的基础和临床意义。申请人通过基因组水平CRISPR-Cas9筛选发现鞘氨醇激酶2（SphK2）是调控HSC自我更新能力加速损伤修复的关键基因之一，并进一步通过SphK2敲除小鼠验证，缺失SphK2不仅使的稳态下HSC的自我更新能力明显加强，且显著增加HSC在化疗损伤后的再生修复以及HSC骨髓移植后的造血重建。这表明SphK2是增强HSC自我更新能力的重要分子靶标。由于已知SphK2可促进细胞核内鞘氨醇-1-磷酸（S1P）的生成进而抑制组蛋白乙酰化，申请人计划利用已建立的痕量细胞转录组和表观遗传组技术深入研究SphK2调控HSC自我更新能力的分子机制，为临床HSC治疗提供理论基础和分子靶标。

本项目前期研究发现鞘氨醇激酶2（SphK2）敲除能够显著增强HSC的自我更新和化疗后的再生功能，骨髓造血重建速度加快，并且化疗后小鼠生存期明显延长。在此基础上，本项目通过转录组数据的比较发现HSC敲除SphK2后代谢信号通路显著改变，特别是低氧反应相关通路显著上调。结合转录组分析和生理实验，我们进一步获得HIF1a-PDK3作为SphK2下游靶点，进行进一步通过基因敲除小鼠的表型验证，我们确认SphK2可以通过HIF1a-PDK3信号通路调控HSC的低氧反应，从而增强HSC的自我更新和再生功能。除此之外，我们还进一步研究了SphK2在HSC衰老中的作用，我们发现敲除SphK2能够逆转HSC衰老相关基因表达。总之，敲除SphK2能够增强HSC的自我更新和损伤再生，并且延缓衰老。因此，本项目的研究结果SphK2信号通路是增强HSC功能关键分子，并且该通路是潜在的延缓HSC衰老重要靶点，这将为HSC治疗和延缓衰老提供重要的理论依据。