HPA轴兴奋对骨髓干细胞的选择性动员效应及其机制研究

In adult organisms, stem cells localized primarily in the bone marrow have the ability to mobilize and differentiate into multiple cell phenotypes in injury. Although the exogenous stem cells play a significant role in the injury treatment, we have to encounter a serious of theoretical and technical bottleneck, such as their expansion, identification and uncertain fate turnover. One current of tissue repair invokes mobilization of these bone marrow stem cells to sites of injury as seed cells. However, the medicine that mobilizes the stem cells may result in adverse reactions among thirty percent of patients. Hence, the exploration by using endogenous repair mechanism to mobilize the stem cells has been paid much attention. Based on our and other studies, we aim to raise the concept of trinitarian hypothalamus-bone marrow-blood mobilization of bone marrow stem cells in the excitation of hypothalamus-pituitary-adrenal (HPA) axis. In this project we propose to further clarify the detailed mobilization courses in HPA axis activation. We will conduct studies in a novel rodent model, a genetically modified CRH deficient mice and chimera mice, which will be succumbed to acute running stress to induce HPA activation. Therefore, we propose the following hypothesis: 1. In response to a single episode of traumatic stress, HPA response and glucocorticoid elevation contribute to the mobilization of bone marrow stem cells at least by modulating the chemotaxis response, favoring the migration toward injured tissues. 2. Prolonged HPA activation and elevated glucocorticoid provoke the coupling of N-formyl peptide receptor (FPR)and CXCR4, which promotes the intracellular Ca2+ mobilization and FAK/paxillin signaling. 3. A persistent modulation of FPR-CXCR4 interactions in HPA response promotes their mobilization and migration toward injury sites essential to functional tissue repair. To test these hypotheses, we propose the following specific aims: 1. Using an animal model of single acute stress, to confirm responses of the bone marrow stem cell mobilization between CRH wild type and CRH knockout mice and the origin of these stem cells in chimera mice. 2. In vitro and in vivo to determine effects of gulcocorticoid on the N-formyl peptide induced chemotaxis in bone marrow stem cells and its relation to FPR and CXCR4. 3. To determine the role of FPR and CXCR4 coupling in the processes of mobilization of bone marrow stem cells. The research concerning the mobilization mechanism of stem cells is of widespread significance for the promotion of clinical treatment with stem cells.

有效动员干细胞并向损伤区募集是干细胞修复受损组织的关键。既往药物动员可引起近1/3的病人出现不良反应。因此，探寻利用内源性修复机制动员干细胞的设想备受关注。本项目在前期发现下丘脑-垂体-肾上腺(HPA)轴兴奋选择性动员骨髓干细胞基础上，提出以下丘脑-骨髓-血三位一体的骨髓干细胞动员概念。研究认为，HPA轴兴奋可通过调节趋化/迁移反应促进骨髓干细胞动员，并向损伤组织靶向归巢。为此，研究在进一步观察HPA轴反应性影响骨髓干细胞动员以及循环干细胞来源验证的基础上，拟获取HPA轴兴奋骨髓干细胞被动员入血，募集到损伤区参与损伤组织修复的分子机制，探讨内源性骨髓干细胞动员的修复学意义。研究旨在初步确立损伤组织修复过程中HPA轴兴奋启动内源性修复的关键点。为通过调控神经内分泌反应启动内源性干细胞修复机制，促进损伤组织功能修复提供新思路。

骨髓源性干/祖细胞(bone marrow stem and progenitor cells, BMSPCs)从骨髓的迁入和移出是作为高度复杂的生物学过程，其作用过程及调控机制尚未深入阐明。有证据显示，运动条件下骨髓的干细胞从骨髓流出并进入血液循环，以参与损伤组修复。本项目研究了下丘脑－垂体－肾上腺(hypothalamic-pituitary-adrenal,HPA)轴在骨髓干/祖细胞动员过程的作用及其调控机制。研究发现，正常小鼠在跑步应激条件下外周血骨髓间充质干细胞和内皮祖细胞数目明显增加，HPA轴功能缺陷的Crh基因敲除小鼠在跑步应激条件下，外周血骨髓间充质干细胞和内皮祖细胞数目增加受到显著抑制，且以上过程与血浆糖皮质激素水平呈正相关关系；为进一步确认糖皮质激素水平和上述血浆干/祖细胞的关系，研究采用微量输液方法，发现补充与上述跑步应激相同水平的糖皮质激素，能够模拟上述骨髓间充质干细胞和内皮祖细胞数目增加反应，并且对Crh基因敲除小鼠，补充糖皮质激素能够有效弥补受损的间充质干细胞和内皮祖细胞动员反应。为排除外周血固有干祖细胞在糖皮质激素作用下数目变化或成熟细胞的去分化反应对上述结果的影响，研究采用上述应激浓度的糖皮质激素体外刺激外周血，发现在相同观察时间内，外周血间充质干细胞和内皮祖细胞数目无变化，因此，应激条件下外周血增加的间充质干细胞和内皮祖细胞可能主要来自骨髓池。同时，体外实验发现，应激浓度的糖皮质激素能够经糖皮质激素受体途径增加间充质干细胞和内皮祖细胞对趋化分子－甲酰肽(FPR)的趋化反应，糖皮质激素受体拮抗剂mifepristone 能够抑制该趋化反应。同时，应激浓度的糖皮质激素能够增加间充质干细胞和内皮祖细胞FPR表达，但抑制其CXCR4表达，且FPR 拮抗剂Cyclosporin H能够显著抑制上述FPR和CXCR4表达调节过程。最后，适度的跑步应激能够增加小鼠伤口愈合率。因此，以上结果提示，糖皮质激素和趋化因子受体FPR协同调节BMSPCs动员反应，其中糖皮质激素以交叉失敏方式调节FPR和CXCR4偶联反应。因此，对HPA反应对FPR-CXCR4相互作用的适度调节有助于BMSPCs向损伤组织迁移并参与修复过程。本课题已按计划完成全部内容，已发表SCI论著1篇（IF: 2.42），中文论著2篇，专利3项，培养硕士研究生1名。