外泌体miR-503在I型胶原免疫调控巨噬细胞极化促进牙周膜干细胞成骨分化中的作用及机制研究

The stability of microenvironment in periodontal tissue plays key roles in the regulation of the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Macrophages play important roles in affecting the inflammatory status and bone metabolism in the periodontal microenvironment. Previous studies have reported that biomaterials based on collagen can maintain the osteogenic or chondrogenic process in inflammatory microenvironment. Our previous study have found that type I collagen (CI), the main organic component of bone, can not only promote tissue repair directly, but also improve the local microenvironment to promote bone repair by immunomodulating the phenotype shift of macrophages in inflammatory environment. In addition, M2 macrophages stimulated by CI can significantly promote the osteogenic differentiation of PDLSCs by transferring exosomal miR-503. So, we speculate that certain components in CI could activate the polarization of M2 macrophages to up-regulate the secretion of exosomal miR-503, which promote the osteogenic differentiation of PDLSCs. Based on previous studies, this study will focus on the following scientific issues: ① the identification of effective components and structure in CI; ② the underlying mechanisms for the polarization of M2 macrophages activated by CI; ③ the roles of exosomal miR-503 from activated macrophages in the regulation of the osteogenic differentiation of PDLSCs. This study will provide a scientific basis for clinical translation of biomaterials based on type I collagen in the periodontal treatments and maintenance.

牙周组织微环境的稳定是调控牙周膜干细胞（PDLSCs）成骨分化的关键，巨噬细胞是牙周微环境中影响牙周炎症及骨代谢的重要因素。已有研究证实，胶原类生物材料能改善炎症环境中的成骨/软骨过程。我们前期研究发现，I型胶原（CI）作为骨组织有机成分的主要物质，能直接促进骨组织修复；还可以通过免疫调控炎性微环境中巨噬细胞的表型改善局部微环境，间接促进骨修复。在CI的作用下，M2型巨噬细胞释放外泌体携带miR-503，显著促进PDLSCs成骨分化。由此推测，CI中的某些成分能调控巨噬细胞的M2极化，上调外泌体miR-503表达，并促进PDLSCs成骨分化。在前期研究基础上，本课题将重点研究以下科学问题：①CI中有效成分及结构的鉴定；②CI促进巨噬细胞M2极化的机制；③外泌体miR-503对PDLSCs成骨分化的作用与机制。这些问题的解决将为基于I型胶原的生物材料在牙周治疗及维护的临床应用提供理论依据。

本课题组以海洋I型胶原（marine type I collagen, MCI）为研究对象，明确MCI在 1mg/ml和3mg/ml工作浓度下，在体外不会影响巨噬细胞的细胞活性。此外，MCI亦不会影响单核/巨噬细胞的炎性趋化功能以及M1型炎性表型。随后，本课题组采用转录组测序的方法，发现MCI可以显著提高M0型巨噬细胞和M1型巨噬细胞高表达趋化因子CXCL13。随后，课题组进一步分离并验证MCI作用下巨噬细胞分泌的外泌体，并证实牙周膜干细胞可以有效吞噬此外泌体。MCI作用下巨噬细胞来源的外泌体内miR-503参与调控牙周膜干细胞的成骨分化。本项目研究可为基于海洋I型胶原的生物材料在牙周治疗及维护的临床应用提供理论依据。