GO促进BMSCs外泌体分泌诱导成骨分化作用的机制研究

Osteogenic induction based on biological materials is always the struggling work in orthopedic biomaterial research. Graphene oxide (GO) is a potential orthopedic biomaterial due to the osteoinductive character to bone mesenchymal stem cells (BMSCs). However, the mechanism of this process is unknown. Our previous study demonstrated that BMSCs seeded on GO membrane completed osteogenic differentiation under non-osteoinductive culture condition. And, the secretion of exosomes and the expression of integrin α2β1 and miR196a increased, and the disturbance of HoxC8-BMP2 pathway can abolish the osteogenic differentiation of BMSCs. Considering the literature reviews and our unpublished results, it’s hypothesized that GO induced osteogenic differentiation of BMSCs may be triggered by activation of integrin α2β1，thereby increasing the secretion of exosomes and the expression of miR196a，which was achieved by Integrinα2β1-ISG/Cortactin-Exosomes-miR196a- HoxC8-BMP2 pathway. To test the hypothesis, we focus on function of miR196a and explore the mechanism of GO induced BMSCs-osteoblast transition in detail through modulating miR196a expression in vitro and in vivo, combined with medication or genetic manipulation of the signaling cascades in this signal pathway. This study is expected to uncover the mechanism of osteogenic differentiation of BMSCs induced by GO, and to provide new insights into osteoinduction biomaterials and reparation of bone defects. This study also holds special hope for providing potential excellent orthopedic biomaterials in the future.

课题组前期研究发现氧化石墨烯（GO）在体外和体内均能够诱导骨髓间充质干细胞（BMSCs）成骨分化，BMSCs源性外泌体分泌增加，integrinα2β1和miR196a表达升高。基于已有研究线索，我们认为GO诱导BMSCs成骨分化可能是激活integrinα2β1，诱导外泌体分泌增加和miR196a表达上调并通过Integrinα2β1-ISG/Cortactin-Exosomes-miR196a-HoxC8-BMP2信号通路实现。为验证该假说，本项目以miR196a为切入点，联合体内外实验通过改变miR196a表达，结合干扰Integrinα2β1-ISG/Cortactin-Exosomes-miR196a-HoxC8-BMP2信号通路上不同位点研究信号传导机制。该项目的实施将揭示GO诱导BMSCs成骨分化的信号传递机制，促进GO骨组织材料临床转化。

骨缺损治疗是临床医生常常面临的一大难题。较大的骨缺损往往难以自行愈合，需要通过骨移植术来重建缺损。自体骨移植是目前临床上治疗骨缺损的金标准，但其存在来源有限及产生供区并发症等缺点。利用骨组织工程技术设计开发新型骨修复材料是未来解决骨缺损治疗难题的重要途径。还原氧化石墨烯（rGO）是石墨烯的主要衍生物，因其具有优异的促成骨特性而在骨组织工程领域受到极大的关注。但rGO诱导骨髓间充质干细胞（BMSCs）成骨分化的具体机制目前尚不明确，极大地限制了rGO相关材料在骨组织工程领域的发展、应用与临床转化。本项目首先构建了rGO薄膜支架材料，并对其促进BMSCs增殖黏附、成骨分化的性质进行了验证；随后将BMSCs与rGO共培养，分离并鉴定上清液中的外泌体（rGO-EXO），明确了rGO-EXO对BMSCs的促成骨分化效应；使用miRNA测序及生物信息学技术对rGO-EXO的促成骨机制进行了研究，使用双荧光素酶报告实验、west blot技术明确了rGO-EXO可以通过miR-26a的高表达抑制BMSCs内TOB1蛋白的表达，最终促进BMSCs成骨分化；同时，我们发现了rGO-EXO在骨修复过程中促进血管生成、调节免疫微环境的效果；最后，我们构建了大鼠颅骨骨缺损模型，明确了rGO-EXO在啮齿类动物模型中促进骨缺损修复的效果。此外，基于前期对二维碳纳米材料（rGO，MXene等）促成骨特性及其具体分子机制的研究，本课题组进一步研发了多种以二维碳纳米材料为基础材料的新型生物材料，并应用于骨缺损修复及骨科相关领域。本项目对rGO材料诱导BMSCs成骨分化的具体机制进行了研究，有利于丰富rGO材料在骨组织工程领域中应用的理论基础，为石墨烯材料在骨组织工程领域的临床转化提供新思路、新途径、新可能。