外源性TGF-β定向诱导内源性干细胞归巢促进骨缺损再生修复及归巢机制

Factor-based tissue engineering bone (TEB) , which inducing endogenous stem cells homing by combining with exogenous growth factor, has become a hotspot in the field of tissue engineering, but the application is still limited by understanding of mechanism underlying stem cells homing inducded by growth factor. Some studies have suggested that all three isoforms of TGF-β can induce endogenous stem cell homing to promote bone regeneration and repair, but the difference of the capacity of all three isoforms of TGF-β inducing stem cell homing and the type of homed cells and homing manchanism remains unclear. Combined with our previous study finding and the latest research progress, we put forward the following hypothesis in this project that TGF-β induces mesenchymal stem cells (MSCs) homing to promote bone regeneration and repair via MCP-1/ CCR2 and TGF-β/TβR two signal axes. This study intends to compare the capacity of all three isoforms of TGF-β inducing MSCs migration in vitro and to analyze the contribution of MCP-1/ CCR2 and TGF-β/TβR two signal axes to MSCs migration by experiments which imitate in vivo condition; and to explore mechanism of TGF-β regulating two signal axes. The implementation of this project will provide experiment evidence for clinical translation of bone tissue engineering and optimizing tissue engineering construction technology.

通过结合外源性生长因子诱导内源性干细胞归巢的Factor-based组织工程骨（TEB），已成为组织工程领域的研究热点，但其应用仍受限于对生长因子诱导干细胞归巢机制的认识。研究提示，体内已知的3种TGF-β均能通过诱导内源性干细胞归巢促进骨再生修复，但3种TGF-β诱导细胞归巢能力的差异、归巢细胞的种类与归巢机制等尚不清楚。结合我们前期课题的研究发现与最新研究进展，本项目提出TGF-β通过MCP-1/CCR2与TGF-β/TβR两条信号轴定向诱导内源性间充质干细胞（MSCs）归巢促进骨再生的假说。拟通过模拟体内环境，比较3种TGF-β在体外诱导MSCs迁移的能力，分析两条信号轴在MSCs诱导迁移中的作用。本项目的实施可为组织工程骨的临床转化以及构筑技术的优化提供实验依据。

本项目依托组织工程国家地方联合工程实验室，在前期实验研究和已有文献报道的基础上，进一步探讨了TGFβ诱导人间充质干细胞（hMSCs）迁移的可能机制，现已完成相关体外实验、数据的统计整理以及SCI论文的撰写工作。. 本项目通过体外实验，观察了3种不同浓度TGFβ诱导MSCs迁移的能力，对比了每种TGFβ对不同组织来源MSCs的诱导差异，分析了TβR/Smad信号通路在TGFβ直接诱导MSCs迁移中的作用。实验发现，三种TGFβ均对脐带Wharton’s Jelly来源的MSCs迁移无直接促进作用，但TGFβ1和TGFβ3却能直接诱导人骨髓来源的MSCs（hBMSCs）迁移，其中25ng/ml的TGFβ1和的TGFβ3具有显著趋化性，且这一趋化性均依赖于TGFβ/Smad途径。此外，我们进一步建立人脐动脉平滑肌细胞（hUASMCs）与hBMSCs的体外共培养体系。在共培养体系中，hUASMCs对TGFβ3诱导hBMSCs迁移有显著促进作用。TGFβ3可刺激hUASMCs 高表达MCP-1，且表达的MCP-1能分泌至培养液中，激活共培养hBMSCs的CCR2受体表达；这一间接调控的MCP-1/CCR2信号途径比TβR/Smad信号途径诱导 hBMSCs迁移的效能更高。本项目初步探讨了TGFβ直接和间接诱导MSCs迁移的可能机制，为阐述TGFβ体内诱导MSCs归巢机制提供了实验依据，也为促进MSCs归巢进而加速骨修复提供一些新的靶点和研究思路。