肠道菌群在胚胎造血干细胞发育中的作用及机制

Though it has been reported that intestinal microbiota affects the function and homeostasis of hematopoietic stem cells in bone marrow both in clinical researched and in animal models, the molecular mechanism of this interaction still remains unclear. Since a variety of the regulatory factors (both endogenic and ectogenic) involved in embryonic hematopoiesis and adult hematopoiesis are similar, embryonic hematopoietic stem cells are supposed to be an ideal model for researches on bone marrow hematopoietic stem cell. In this projest, we were planning to investigate how the intestinal microbiota affects the embryonic hematopoiesis by using germ-free (GF) zebrafish. We would focus on the investigating of the molecular mechanism by analysing the changes occurs in ths GF zebrafish model on the related signaling pathways (such as TLR/myd88 and NOD1), the formation and function of the immunocytes, as well as the cytokines that might be invovled, followed by the transcriptomic analysis based on the RNA-seq results to screen for more potentially involves pathways. Strains or metabolins that are able to induce the development of hematopoietic stem cells would be also screened from the fecal microbiota pools of both human and zebrafish by using our GF zebrafish model. Furthermore, epigenetic regulators (such as gene chd7) that might be involved in the interaction between intestinal microbiota and hematopoiesis would also be analysed. Thus, the results that could been generated from this project may provide new strategies for the development of clinical treatments on graft-versus-host disease after allogeneic hematopoietic stem cell transplantation through regulating intestinal microbiota homeostasis.

近年来，临床和小鼠模型研究都证实肠道菌群对骨髓造血干细胞的功能和稳态产生显著影响，但两者之间的相互作用关系和机制尚不明确。胚胎中造血干细胞的发育是研究骨髓造血干细胞的良好替代模型。其发育过程受到众多内外因子调控的影响与骨髓造血干细胞相似。在本项目中，我们提出以斑马鱼为模式生物，建立无菌胚胎培养方式，分析肠道菌群对造血干细胞发育的影响。通过分析TLR/myd88,NOD1等信号通路、免疫细胞的形态和功能以及细胞因子在无菌培养下的改变来研究机制，并进一步通过对比转录组的差异来筛选其它作用分子。利用无菌斑马胚胎，本项目还将鉴定不同的斑马鱼肠道和人肠道菌种及代谢物对造血干细胞发育的调控和选出对干细胞生长促进的菌种或代谢物。此外，本项目还将分析表观遗传学修饰基因如chd7是否参与肠道菌圈对造血干细胞的调节。本项目的研究结果将为临床上调控肠道菌群的平衡与造血干细胞移植相关的治疗提供新的理论依据。

目前在微生物-宿主相互作用的相关研究中，对于共生菌与宿主造血功能调控之间相互作用的研究十分有限。在本研究中，我们以斑马鱼为模式生物，首先通过构建野生型斑马鱼无菌胚胎的培养模型，发现正常肠道菌群的缺失会导致胚胎造血干细胞数量的减少，且这些表型可通过菌群的重定植恢复。证明正常肠道菌群的存在对于维持斑马鱼胚胎血干细胞的正常发育是必要的。我们还进一步证明，肠道菌群可以在胚胎发育过程中通过调节炎症因子的表达，对宿主胚胎造血干细胞的发育进行调控。随后，我们通过对chd8-/-斑马鱼系中肠道菌群失衡和造血干细胞发育异常之间相互关系的研究，验证了肠道菌群失衡与宿主造血干细胞发育异常之间的关联性，且证实通过对肠道菌群失衡状态的矫正，可以在一定程度上使异常的造血干细胞发育得到恢复。此外，本研究通过对菌群中分离出的单菌进行功能验证，发现不同的菌株对于同一宿主的可能存在不同的调控作用，甚至同一菌株在不同宿主中的调控作用也可能不同。我们最终还筛选出了一株可以在一定程度上恢复chd8-/-斑马鱼胚胎中异常的造血干细胞发育表型的菌株（BHI1-3）。本研究中的研究结果以及筛选出的菌株或许将为临床上调控肠道菌群的平衡与造血干细胞功能异常疾病相关的治疗提供新的理论依据。