急性移植物抗宿主病造血异常及造血微环境重塑的机制研究

Through a haplo-major histocompatibility complex matched acute graft-versus-host disease (aGVHD) murine transplantation model, we have observed progressively suppression of donor-derived hematopoiesis and serious blockage of differentiation from common myeloid progenitors toward megakaryocyte/erythrocyte progenitors during aGVHD development. We found that donor-derived hematopoietic stem cell (HSC) potential recovered after being taken out of the aGVHD host environment, suggesting that aGVHD hematopoietic niche plays a crucial role in aGVHD hematopoietic failure. We found that the adipocytes were significantly reduced in the bone marrow hematopoietic microenvironment of both aGVHD patients and aGVHD mouse model. However, the process how aGVHD remodels the niche, which in turn interrupts the function of donor-derived HSCs and hematopoietic progenitor cells (HPCs) is still an important and clinically relevant question that deserves a thorough investigation. We aim to elucidate the molecular mechanism and suppressive effect of hematopoietic niche remodeling under aGVHD pathological condition, to study the role of key cells and factors in the process of aGVHD development and to explore the role of mitochondrial transfer and exosome transfer in aGVHD hematopoietic microenvironment. We also intend to explore the intrinsic mechanisms on how aGVHD suppresses donor-derived HSCs and HPCs during aGVHD development, including genetic and epigenetic aspects. This study is aimed to discover novel molecular targets and establish better strategies to improve the current therapeutic effect of aGVHD.

本课题组前期构建半相合急性移植物抗宿主病（aGVHD）小鼠模型发现供者来源造血系统受到显著抑制，髓系祖细胞（CMP）向巨核系红系祖细胞（MEP）分化受阻。脱离aGVHD环境，供者来源造血干细胞（HSC）潜能得以恢复，提示aGVHD造血微环境对造血衰竭起主要影响作用。在aGVHD的小鼠模型中，骨髓造血微环境的脂肪细胞明显减少。aGVHD如何重塑造血微环境以及进一步影响供者来源正常造血及其作用机制，目前知之甚少。本课题旨在通过研究aGVHD条件下造血微环境重塑的分子机制，寻找微环境中重要细胞及因子在aGVHD疾病发展过程中的作用以及变化规律，探索线粒体及外泌体转移在其中发挥的作用，并继而抑制正常造血的机理。同时分析受抑的供者来源正常造血干/祖细胞的遗传与表观遗传调控的交互作用机制。本课题的开展，可进一步阐明aGVHD微环境对供者来源造血细胞的影响机制，探索提高aGVHD治疗效果的新靶点和策略。

严重的急性移植物抗宿主病（aGVHD）导致恶性血液病患者在异基因造血干细胞移植（HSCT）后预后不良。aGVHD发病期间，患者的造血功能逐渐受到抑制，而供体来源的造血干细胞的造血再生潜能保持完整。因此，骨髓干细胞生存的微环境（niche）被认为可能是aGVHD的主要目标。我们重点通过单倍体MHC匹配的小鼠骨髓移植模型研究了造血微环境中的关键成分，包括间充质干细胞（MSC）、成骨细胞和脂肪细胞。通过非造血骨髓细胞的单细胞RNA测序（scRNA seq）技术，我们发现在aGVHD发病过程中，MSC的脂肪生成和成骨细胞生成相关的关键调控基因的表达显著下调。小鼠和人骨髓微环境细胞的体外培养也显示了来自aGVHD疾病状态下骨髓造血微环境中的MSC的分化潜能缺陷。通过比较对照组和aGVHD荧光标记转基因小鼠股骨的荧光共聚焦图像，我们发现，在aGVHD造血微环境中，骨髓间充质干细胞、成骨细胞和脂肪细胞均显著减少。基因火山图和定量mRNA表达测试证实，aGVHD小鼠骨髓间充质干细胞中与脂肪细胞和成骨细胞谱系相关的基因显著下调。通过给aGVHD小鼠分别予芦可替尼和MSCs进行干预，我们发现芦可替尼能够部分抵消aGVHD的造血微环境被破坏的情况，并为供体来源的造血细胞提供更有利的微环境，而MSCs干预下确实可以减轻aGVHD的严重程度并延长aGVHD小鼠的生存期，但骨髓造血微环境未得到改善。通过使用芦可替尼预刺激MSCs后MSC体外扩增培养结果显示，经芦可替尼预处理的MSCs不仅能其体外增殖能力得以提高，而且aGVHD小鼠的存活时间得到进一步延长，提示芦可替尼可能是HSCT治疗过程中骨髓MSCs体外扩增的理想预处理药物。这些数据为aGVHD的发病机制提供了新的见解，并可能改善该病的临床治疗提供了新的思路。