IGFBP3在蛋白组织工程骨募集宿主MSCs参与骨形成过程中的作用机制研究

Tissue engineering bone (TEB) is an important strategy for large bone-defect repair, and protein-based tissue engineering bone (PTEB) could recruit endogenous mesenchymal stem cells (MSCs) for bone regeneration, the mechanism in which is not clear. Our previous study showed that the expression of insulin-like growth factor binding protein 3(IGFBP3) was strongly elevated in PTEB compared to the control scaffold, and the expression of transforming growth factor beta receptor II(TβRII) was up-regulated significantly during IGFBP3 promoted the migration of MSCs. The hypothesis is put forward that IGFBP3 directly actives TβRII/Smad, and IGFBP3/TβRII/Smad pathway would play an important role on PTEB-induced recruitment of endogenous MSCs for bone regeneration. The study firstly intends to demonstrate the interaction between IGFBP3 and TβRII. The the role of IGFBP3/TβRII/Smad pathway on MSCs migration and cell cytoskeleton rearrangement would be analyzed. Secondly, the effects of IGFBP3 signaling pathway on migration of MSCs would be evaluated in vitro to clarify the mechanism of MSCs migration induced by PTEB. The bone defect model in SCID mice was adopted to test the expression of signaling pathway and estimate the role of IGFBP3 on PTEB-induced recruitment of endogenous MSCs for bone regeneration. This implementation of the project will provide experimental data to illuminate the mechanism of endogenous stem cell recruitment induced by PTEB.

组织工程骨(TEB)是修复大段骨缺损的重要策略，蛋白组织工程骨(PTEB) 能募集宿主MSCs参与骨形成，但作用机制不明。我们前期实验证实，PTEB高表达的IGFBP3在促进MSCs迁移时上调TβRII表达。结合文献分析，我们推测IGFBP3以直接激活TβRII/Smad途径的方式在PTEB募集MSCs参与骨形成过程中发挥重要作用。为此，本项目拟通过体外实验研究IGFBP3与TβRII的相互作用、分析IGFBP3/TβRII/Smad通路在MSCs迁移和细胞骨架重排中的作用；采用PTEB和MSCs共培养模型，经阻断实验，观察IGFBP3通路在PTEB促进MSCs迁移中的作用，阐明PTEB诱导MSCs迁移机制；最后采用骨缺损移植模型，检测移植PTEB前后该通路分子在损伤处的表达变化，评价IGFBP3的阻断对PTEB募集宿主MSCs及成骨能力的影响，为阐明PTEB募集机制提供实验依据。

组织工程骨(TEB)是修复大段骨缺损的重要策略，蛋白组织工程骨(PTEB) 能募集宿主MSCs参与骨形成，但作用机制不明。蛋白芯片结果证实PTEB中IGFBP3高表达，提示IGFBP3可能在PTEB募集成骨中发挥重要作用。我们通过全转录组测序对PTEB体内募集细胞进行检测并筛选到8个高表达迁移受体，qPCR检测进一步筛选到IGFBP3能上调hBMSC中TβRI及CCR2表达。免疫共沉淀证实IGFBP3可以结合TβRI及CCR2两个迁移受体。qPCR、Western blot证实IGFBP3以激活TβRI/II-Smad3途径及CCR2途径的方式促进hBMSCs 迁移。利用shRNA沉默技术制备低IGFBP3表达的PTEB，结合流式细胞术、免疫荧光、Transwell、 Micro CT以及HE染色等实验证实IGFBP3信号途径在PTEB促进hMSCs迁移和募集体内MSCs参与骨形成过程中发挥主要作用。IGFBP3是一个重要的诱导MSCs归巢的分子而IGFBP3通路为PTEB募集成骨主要通路。本项目接收和发表SCI文章共4篇及申请发明专利1项、授权专利1项。