LPS刺激牙髓干细胞来源的外泌体miRNAs介导根尖周炎免疫调节和骨吸收的作用研究

In apical periodontitis, oral pathogens (mainly infected dental pulp) provoke inflammatory and immune response in the apical area that induce periradicular tissues destruction and bone resorptive lesions. Exosomes are extracellular vesicles secreted from various cell types and contain many microRNAs (miRNAs). Increasing evidence has suggested that mesenchymal stem cells-derived exosomes play a pivotal role in variety of physiological and psychological processes such as immune defense, inflammation，osteogenesis and angiogenesis. For the first time, we found that dental pulp cells derived-exosomes with highly angiogenic potential for possible use in pulp regeneration. Furthermore, miRNAs expressions referred to inflammation and immunity were regulated in exosomes that secreted from LPS-preconditioned dental pulp stem cells, which reveals it may associated with immune response, bone resorption of apical periodontitis and thus improve tissue repair and regeneration. Based on these work, we used miRNA microarray and bioinformatics analysis to investigate the expression profiles of miRNAs and the potential target genes in LPS-Exo. Then, the role of LPS-exo in macrophage polarization and osteoclast differentiation and signaling pathways underlying this process were explored. Furthermore, a rat model of apical periodontitis will established by injecting the inhibitor of exosomes. MicroCT and immunochemical staining were performed to observe the inflammation and bone resorption in apical tissues. These will contribute to deepening our understanding of the molecular mechanisms of immunomodulation and bone resorption, and offer theoretical basis to prevent and treat apical periodontitis and promote tissue repair and regeneration in future.

根尖周炎是主要由感染牙髓所导致的根尖周组织损坏和骨吸收的局部炎症免疫反应。外泌体（Exosomes）是由各种细胞分泌的包裹有miRNAs等生物活性物质的胞外囊泡。间充质干细胞外泌体参与免疫应答、炎症反应、骨重建和血管新生等重要的生理病理过程。我们首次发现牙髓细胞外泌体发挥促血管新生潜能，既往文献中炎症免疫相关miRNAs在脂多糖刺激牙髓干细胞后外泌体（LPS-Exo）中的表达发生变化，提示LPS-Exo miRNAs可能参与根尖周炎的炎症免疫调控和骨吸收，从而影响组织的修复再生。本项目拟通过miRNAs芯片和生物信息学分析LPS-Exo miRNAs表达谱的变化及靶基因；LPS-Exo对巨噬细胞免疫调节和破骨细胞分化的影响及相关miRNAs调控机制；体内实验阻断外泌体后观察根尖周炎症免疫反应和骨吸收情况。这将充实根尖周炎的分子调控机制，为预防和治疗根尖周炎及促进组织再生修复提供理论依据。

根尖周炎引起局部炎症免疫反应，导致根尖周组织破坏和骨吸收。研究发现，间充质干细胞（MSCs）来源的小细胞外囊泡（MSC-sEV）/外泌体（Exosomes，Exos）参与免疫应答、炎症反应、血管新生和骨重建等重要的生理病理过程。课题组前期研究发现牙髓细胞外泌体的促血管生成潜能。基于此，本项目首先利用单细胞转录组学解析根尖周炎病变组织中各细胞类型及细胞之间的相互关系，并探索根尖致病菌对巨噬细胞发生泛凋亡（PANoptosis）的调控作用；其次探讨了牙髓干细胞（DPSCs）来源外泌体和小细胞外囊泡（sEV）在血管生成以及免疫调节和骨吸收中的作用。最后探讨了丝素蛋白/聚乙醇酸（SF/PGA）支架材料在骨组织工程中的作用。结果如下：（1）揭示根尖周炎病变组织单细胞表达图谱，获取4种免疫细胞和4种非免疫细胞，其中非免疫细胞群的基因表型变化与细胞的转化及状态受免疫细胞调控，决定根尖周炎的转归；粪肠球菌感染巨噬细胞诱导发生PANoptosis过程中存在细胞的自身调控，抑制caspase-1活化可同时抑制PANoptosis中焦亡和凋亡途径，减少细胞死亡。（2）低氧DPSCs外泌体通过转运LOXL2发挥促血管生成作用；低氧可促进DPSCs释放sEV，改变miRNAs表达谱，经由miR-210-3p/NF-kB1信号通路诱导巨噬细胞M2向极化并抑制破骨细胞生成，有效减轻LPS诱导的炎性骨吸收；（3）SF/PGA 复合材料大孔径支架更有利于体内外成骨。上述结果为了解根尖周炎的分子调控机制，预防和治疗根尖周炎骨吸收及促进组织再生修复提供理论依据。