IGFBP3/NOD1/OSM信号途径在巨噬细胞参与基质依赖型组织工程骨成骨中的作用机制研究

Matrix-based tissue engineering bone (MTEB) is an important strategy for large bone-defect repair, and instructs mesenchymal stem cells (MSCs) to bone regeneration mediated by macrophage(Mp), but 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 nucleotide-binding oligomerisation domain 1(NOD1) was up-regulated significantly during IGFBP3 induced the secrection of oncostatin M(OSM) from Mp. The hypothesis is put forward that IGFBP3 directly actives NOD1, and IGFBP3/ NOD1 pathway would play an important role on the MTEB-upregulated the secrection of OSM from Mp, which could promote osteogenesis of endogenous MSCs for bone regeneration. The study firstly intends to demonstrate the interaction between IGFBP3 and NOD1. The role of the block of NOD1 pathway on the IGFBP3-induced the secrection of OSM from Mp would be analyzed. Secondly, the MTEB with Igfbp3 knock-down would be prepared; the co-culture medium of MTEB and Mp would be used to incubate MSC and the role of IGFBP3/ NOD1/OSM pathway on the osteogenesis of MSC enhanced by MTEB would be evaluated. At last, the bone defect model in SCID mice would be adopted to estimate the role of IGFBP3 on MTEB-induced osteogenesis of endogenous MSC for bone regeneration. This implementation of the project will provide experimental data to illuminate the mechanism of MTEB-induced endogenous MSC osteogenesis mediated by Mp.

基质依赖型组织工程骨(MTEB)是修复大段骨缺损的重要策略，可经Mp的介导加速MSC成骨，但作用机制不明。前期实验结果显示，MTEB中高表达的IGFBP3，在诱导Mp分泌OSM以促进MSC成骨分化时，显著上调Mp中NOD1表达。结合文献分析，我们推测IGFBP3以直接激活NOD1途径的方式在MTEB刺激Mp分泌OSM以促进宿主MSC成骨中发挥重要作用。为此，本项目拟通过体外实验研究IGFBP3与NOD1的相互作用，分析NOD1基因的沉默及其途径的阻断对IGFBP3诱导Mp分泌OSM的影响；利用RNA干扰技术制备IGFBP3沉默的MTEB，观察MTEB和Mp的共培养液促进MSC成骨分化的情况，明确IGFBP3途径在MTEB促进MSC成骨分化中的作用；最后采用骨缺损模型，考察IGFBP3的阻断对MTEB促进宿主MSC分化成骨的影响，为阐明Mp参与MTEB促MSC成骨的机制提供实验依据。

基质依赖型组织工程骨(MTEB)是修复大段骨缺损的重要策略。MTEB可能经巨噬细胞介导加速MSC成骨，但作用机制不明。质谱结果证实MTEB中TGFβI和MIF高表达，提示TGFβI和MIF可能在巨噬细胞介导MTEB成骨中发挥重要作用。我们通过clodronate liposomes制备的巨噬细胞耗竭的SCID鼠股骨偏心性缺损模型，以及体外支架材料-巨噬细胞-hBMSCs共培养模型，证实巨噬细胞介导了MTEB体外内骨形成。进一步地，流式细胞术证实MTEB植入体内骨缺损处3天，能促进宿主M2型巨噬细胞募集而减少M1型巨噬细胞聚集。在体外Transwell迁移模型中，MTEB能促进M2型巨噬细胞迁移率增加62.52 ± 8.06%；通过qPCR检测M2型巨噬细胞标志性markers CD206和CD163显示MTEB能促进M1型巨噬细胞向M2型再极化；促进抗炎因子抑制促炎因子的表达。二代测序分析MTEB对招募巨噬细胞的信号通路，结合慢病毒转染制备了TGFβI敲低的MTEB，经qPCR和western blot的体外验证，证实MTEB以TGFβI依赖的形式调控涉及ITGβ7, ITGα9和ITGβ1的多个整合素信号途径促进更多M2型巨噬细胞的累积，从而减少炎性，以致MTEB支架在植入SCID鼠的偏心性骨缺损模型后4周，其表面的纤维囊厚度减少至基础支架的1/10，骨量增加了10倍。另一方面，利用全转录组测序，结合qPCR和Western blot验证等方法，证实MTEB显著促进hBMSCs中BMP2、OSM、FGF2、TGFβ3等促成骨细胞因子的表达以及CD74/CD44信号途径的关键分子的表达。而MTEB刺激巨噬细胞的条件培养基能促进hBMSCs的ALP表达，茜素红矿化水平以及成骨分化基因（OCN和OSX）表达；利用慢病毒转染制备了mif敲低的MTEB(shmif MTEB)进行反向验证，MTEB中mif降低可以下调巨噬细胞的成骨因子OSM、FGF2、TGFβ3表达，抑制CD74信号途径的下游p38的磷酸化；其条件培养基也抑制了hBMSCs的成骨分化能力，以及shmif MTEB体内成骨性能显著下降；证实MIF/CD74信号途径在巨噬细胞介导的hBMSCs体外成骨分化和体内MTEB骨形成中起主要作用。本项目发表SCI文章共2篇及获得发明专利1项授权。

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