Nell-1调控BMSCs外泌体中成骨相关miRNAs在种植体骨结合中的作用及机制研究

Nell-1 has positive and specific effects on bone formation. Exosomes regulate the gene network of receptor cells through transferring the active substances secreted from cells. Researches shows that BMSCs exosomes mediate the signaling pathways to promote osteogenesis by carrying osteogenesis related miRNAs. Our former study found that the overexpression of Nell-1 can promote BMSCs osteogenic differentiation and increase expression of miR-21，miR-26a, and miR-218 in exosomes on hydrophilic titanium surface. We hypothesize that Nell-1 regulates osteogenesis related miRNAs in BMSCs exosomes to promote bone formation by adjusting the osteoblast/osteoclast signaling pathways during implant osseointegration. This project examine the differential expression of miRNAs in exosomes of Nell -1 overexpression BMSCs (BMSCs-Nell-1)，screen out the downstream target genes and analyze the interaction of the proteins with DNA as well as proteins with RNA, also verify the effects of BMSCs-Nell-1 exosomes to implant osseointegration in vivo. This project establishes theoretical support and experimental foundation for illumination the molecular regulation mechanism of BMSCs osteogenic differentiation and promotion of titanium implant osseointegration.

Nell-1具有高效特异的促成骨作用。外泌体通过转运细胞分泌的活性物质改变受体细胞的基因调控网络。研究表明BMSCs外泌体通过携带成骨相关miRNAs介导成骨信号通路来促进成骨。本课题组前期研究发现，在活性亲水钛片表面，Nell-1过表达的BMSCs外泌体可促进BMSCs成骨向分化，且外泌体中miR21、miR26a、miR218的表达显著上调。我们提出在种植体骨结合过程中，Nell-1是通过调控BMSCs外泌体中的成骨相关miRNAs，介导成骨／破骨信号通路来促进成骨的设想。本课题拟通过检测Nell-1过表达的BMSCs外泌体miRNAs的差异表达谱，筛选下游靶基因，分析其蛋白与DNA及蛋白与RNA的相互作用，并体内验证Nell-1过表达的BMSCs外泌体对种植体骨结合的影响，为阐明间充质干细胞成骨向分化的分子调控机制、促进种植体骨结合提供实验基础与理论依据。

基于骨髓间充质干细胞的移植和种植体表面修饰可以有效地再生组织和骨整合。但是，干细胞凋亡、的免疫排斥和致畸形等阻碍了其应用。细胞外囊泡 (EVs) 介导的无细胞治疗是一种有前景的干细胞移植的替代方案。已知Nell1分子具有高效特异的促成骨作用，但尚不清楚EVs是否参与了这一过程。本项目研究表明，Nell1是通过调控BMSCs细胞外囊泡中miR-25-5p的表达，促进SMAD/ERK信号通路的激活，进而促进成骨。3D-Nell1/EV-水凝胶系统可有效实现体内的面积骨缺损修复。此外，本项目还发现，BMSC-EV可用于种植体表面修饰，可通过激活RhoA/ROCK信号通路来促进钛片表面BMSCs的粘附、扩散和成骨分化。本项目发现的miR-25-5p-SMAD2 信号轴的扩展了对于Nell1成骨诱导能力的认知，显示了Nell1/EVs作为一种新型无细胞骨再生策略的潜力。提出了EVs在钛表面可改变细胞生物学行为的新见解，提供种植体表面修饰的新途径。