创伤后Slit2/Robo4信号通路诱导内皮祖细胞定向分化的作用机制研究

Lack of regeneration of endothelial progenitor cell (EPC) and impaired angiogenesis would contribute to progression of multiple organ dysfunction syndrome (MODS) caused by trauma. Slit2/Robo4 is an endothelial cell-specific signal pathway to induce directional differentiation of EPC which would promote angiogenesis and maintain vessel integrity. But the mechanism of effects of Slit2/Robo4 on EPC during the phase of MODS after trauma remains unknown. In our former study, we established the MODS animal model and the methods of cultivation of EPC in vitro successfully. And we would investigate the relationship between the change of capillary densities of important organs and vessel integrity and the expression of Slit2/Robo4 in EPC on our animal trauma model. And we further investigate the expression of VE-cadherin and UNC5B (a vascular Netrin receptor) to illustrate the mechanism of the directional differentiation of EPC induced by Slit2/Robo4 in vitro whether the calcium channel or UNC5B receptor was involved. The present study, for the first time investigate the mechanism of directional differentiation of EPC induced by Slit2/Robo4 during the phase of MODS after trauma, to explore a novel cell-based, vascular endothelium-targeted therapeutic strategy for MODS.

内皮祖细胞（EPC）是参与机体创伤修复的重要细胞，与创伤预后密切相关；Slit2/Robo4信号转导通路可诱导EPC定向分化为成熟血管内皮细胞并促进血管新生及维持血管内皮稳定性，最大限度增强EPC的修复作用，但创伤后Slit2/Robo4表达强度变化及其对EPC的调控机制尚不清楚。本项目利用我们已成功建立的动物创伤模型和EPC体外培养体系，首先观察创伤后机体血管新生及血管内皮稳定性的改变和EPC内Slit2/Robo4表达水平变化之间的相关性；进一步在体外EPC培养体系中，研究Slit2/Robo4对EPC的定向分化及其介导的血管成形和血管内皮稳定性功能的调控作用，并探讨其内在机制是否涉及钙离子通道或UNC5B受体。旨在阐明创伤进程中Slit2/Robo4信号转导通路诱导EPC的定向分化并促血管新生功能及维持血管内皮稳定的具体调控机制，为临床防治创伤后MODS探索新的治疗靶点提供理论依据。

本课题首先在前期研究的基础上进一步完善了小鼠源性内皮祖细胞的分离和培养体系。并在和南方模式动物公司联合构建了Robo4-/-敲除小鼠的基础上，利用盲肠穿孔复制出小鼠创伤后MODS模型。进一步检测在创伤后MODS进展过程中内皮祖细胞及Robo4，UNC5B受体的表达变化。最后在体外内皮祖细胞的培养体系中，探讨Robo4对内皮祖细胞定向分化功能的影响及其相关机制。研究发现：脓毒症发展过程中，内皮祖细胞的数量迅速上升，在MODS发生前达到峰值，随后出现明显下降，而Robo4表达水平则逐渐升高，至脓毒症中期达到峰值，随后逐渐下降，二者在脓毒症引起的MODS进展过程中的变化成正相关。Robo4+/-、Robo4-/-和WT三种基因来源的EPC的细胞形态和鉴定无明显差别，Robo4-/-敲除小鼠的血管形成能力和迁徙功能明显下降。在EPC的体外培养体系中，外源性的Slit2可以诱导Robo4及其下游VE-cadherin的表达升高，从而提高EPC的定向分化能力。研究证明Slit2／Robo4可以在病态内环境中最大程度提高EPC的定向分化能力，为临床脓毒症后MODS的防治提供的新的思路及理论实验基础。