骨组织损伤后募集MSC定向迁移修复的趋化因素SDF-1的来源及其Epac-Rap-Rac/Arpin作用通路的机制研究

Tissue-engineered bone (TEB) is one of promising methods for repairing huge bone defect. TEB could recruit mesenchymal stem cells (MSC) to repair bone defect by injury with transplantation and seeding cells’ osteoinductive,in which MSC mobilization and migration are involved. SDF-1 is a important chemotactic factor and is involved in MSC mobilization and migration. However,the source of SDF-1 and the mechanism of SDF-1 on MSC migration are not clear. SDF-1 is produced and secreted by many kinds of cells. The different sources possiblly co-work together for MSC, which need be further determined. The pathway of Rac-Arpin has been found recently, and that effects on directional migration of MSC. Our pre-experiment also showed that Arpin was expressed in MSC, and was regulated by SDF-1. Recently, the effect of Epac-Rap-Rac on MSC migration was found, but the action of SDF-1 is not clear during this process. Based on these, the hypothesis was logically set up that Epac-Rap-Rac/Arpin pathway meidates MSC migration induced by SDF-1 during bone injury and repair. Firstly, We would detecte which source of SDF-1 in the different tissue is important for MSC migration by knock-out mice. The SDF-1 originated from MSC, endothelial cell, osteoblast, osteocyte and the whole body are knock-out respectively, and their effect on MSC migration are assayed. At different gene point, including Epac,Rap-1,Rac,and Arpin,the knock-out and kock-in mice are set up, the effect of Epac-Rap-Rac/Arpin pathway on MSC migration will be evaluated on the basis of these experiments. This study will make the mechanism of MSC migration clearer during bone injury and repair, supply a guide for elevating osteogenic potency of TEB, and explore a new targeted point and strategy of increasing MSC migration during application TEB.

MSC的定向迁移是组织工程骨治疗骨缺损最重要的机制之一，虽然发现SDF-1是重要的趋化因素，但是SDF-1的来源和其作用通路并不明确，影响相关机制在骨修复重建中的应用。体内多种组织和细胞可以产生SDF-1，对于MSC的迁移可能作用效果不一，需要进行研究。Rac和Arpin环路作为调节细胞定向迁移的通路，新近被发现；课题组也发现Arpin可在MSC内高表达并显著受到SDF-1的作用调节；而Epac-Rap-Rac在MSC迁移中的作用近来也被发现，但与SDF-1作用是否相关不明。基于上述，提出“骨组织损伤后，宿主体内的SDF-1是通过Epac、Rap-1、Rac以及Arpin通路来定向募集体内MSC迁移”。通过不同细胞内敲除SDF-1来观察募集MSC迁移的SDF-1的来源；通过不同基因水平的正向增强和负向敲除或减弱来检查上述假说及相应的作用机制。为进一步募集MSC提供新的作用靶点和更好的效果。

间充质干细胞（MSC）的定向迁移是组织工程骨（TEB）治疗骨缺损最重要的机制之一，虽然发现SDF-1是重要的趋化因子，但是SDF-1的来源和其作用通路并不明确，无法利用相关机制提升骨修复重建效果。因此，本研究的主要内容包括两方面：一是SDF-1激活MSC定向迁移的细胞内调控机制；二是组织工程骨局部微环境内SDF-1的来源及其调控机制。基于上述研究内容，本研究首先证实在TEB移植区及骨髓和外周血内SDF-1存在动态的变化，这与MSC由骨髓到外周血向TEB定向迁移相吻合。体内、外证实Epac-Rap-Rac-Arpin轴在SDF-1调控MSC迁移中发挥重要作用，干扰此轴，可显著减少TEB内迁移的MSC的数量，并最终影响局部TEB的骨修复重建能力。其次，本研究通过TEB转录组和单细胞测序分析，发现不同时间、不同细胞内的Epac、Rap-1、Rac-1和Arpin存在动态变化。术后3天高于14天，提示迁移在移植早期即开始；TEB高于DBM，提示TEB组内MSC相关基因被调控更明显，这与TEB组织内MSC数量也更多相一致。而MSC中SDF-1基因表达则术后14天明显高于3天，TEB组高于DBM组；内皮细胞（EC）则相反，术后3天高于14天，而成骨祖细胞（Osx阳性）和成骨细胞（Osteocalcin阳性）表达量较低，几乎检测不到，提示MSC和内皮细胞是TEB中SDF-1的主要来源。相应的TEB组中MSC数量明显多于DBM组。最后，蛋白质芯片定量和单细胞测序分析发现，与未敲除的野生型相反，敲除品系中，大部分品系中SDF-1浓度都是7d低于3d，表明敲除后SDF-1的分泌确实受到严重影响。对比不同敲除品系中SDF-1受影响程度，间充质敲除（P品系）在TEB处的浓度最低，表明间充质细胞敲除了SDF-1后对缺损处SDF-1的影响最大。敲除小鼠对成骨影响从强到弱依次为间充质、成骨、前成骨和内皮。不同品系中，敲除SDF-1基因后，对各自组内MSC中的Epac、Rap-1、Rac以及Arpin的基因表达无明显影响。本研究证实Epac-Rap-Rac-Arpin轴在SDF-1募集MSC向TEB迁移过程中发挥重要作用，而MSC自分泌的SDF-1是TEB中的主要来源，存在自我募集效应，在未来构建TEB时可早期应用SDF-1促进MSC的募集，可通过增强作用轴的活性加速MSC迁移，进而加速骨重建。