机械力诱导的牙周膜干细胞外泌体通过miR-21促进正畸牙根吸收的机制研究

Root resorption is a common complication in orthodontic treatment. Exploring its molecular mechanism has important guiding significance in reducing root resorption in clinical situations. We previously found that the exosomes secreted by periodontal ligament stem cells may play an important role in root resorption and bone metabolism during orthodontic stress. The exosomes of periodontal ligament stem cells induced by mechanical force can promote the expression of osteoclast differentiation factor. At the same time, the exosome-enriched miR-21 is a microRNA that plays an important role in the process of osteoclast. Accordingly, we hypothesized that mechanical force-induced periodontal ligament stem cell exosomes could further promote root resorption in orthodontic teeth through miR-21. This application is to establish a model of mechanical force-induced periodontal ligament stem cells and screen the exosome-specific microRNA, then study the effect of exosome and its delivery of miR-21 on the differentiation of osteoclasts under mechanical force, and finally give the rat model of exosome excitation inhibitor and selective transfection to explore the mechanism of exosome miR-21 involved in regulating root resorption. In conclusion, this study will explore the effect of mechanical force-induced periodontal ligament stem cell exosomes on root resorption in orthodontic teeth through miR-21, so as to deepen the understanding of root resorption mechanism in orthodontic teeth, and provide new ideas for further prevention of root resorption in clinical orthodontics.

牙根吸收是正畸治疗常见的并发症，探讨其分子机制对正畸临床减轻牙根吸收具有重要指导意义。我们前期发现正畸加力时，牙根吸收骨代谢微环境中牙周膜干细胞分泌的外泌体可能发挥重要作用：机械力诱导的牙周膜干细胞外泌体可以促进破骨分化因子的表达，同时外泌体中富集的miR-21是一种在破骨进程中有重要作用的MicroRNA。据此我们提出机械力诱导的牙周膜干细胞外泌体通过miR-21进一步促进正畸牙根吸收的假设。本申请拟首先建立牙周膜干细胞加力模型并筛选外泌体特异性MicroRNA，随后研究机械力作用下外泌体及其递送的miR-21对破骨细胞分化的影响，最终给予大鼠加力模型外泌体激动、抑制剂及选择性转染，探讨外泌体miR-21参与调控牙根吸收的机制。综上，本研究将探讨机械力诱导的牙周膜干细胞外泌体通过miR-21介导正畸牙根吸收的作用，加深对正畸牙根吸收机制的理解，为进一步预防临床正畸牙根吸收提供新思路。

牙根吸收是正畸治疗常见的并发症，探讨其分子机制对正畸临床减轻牙根吸收具有重要指导意义。我们前期发现正畸加力时，牙根吸收骨代谢微环境中牙周膜干细胞分泌的外泌体可能发挥重要作用：机械力诱导的牙周膜干细胞外泌体可以促进破骨分化因子的表达，同时外泌体中富集的miR-21是一种在破骨进程中有重要作用的MicroRNA。据此提出机械力诱导的牙周膜干细胞外泌体通过miR-21进一步促进正畸牙根吸收的假设。. 本项目中，使用经典试剂盒方法在牙周组织的局部免疫微环境中培养PDLSC，并在机械加力后提取出外泌体。对其形态学、分子表型、粒径分布和miRNA含量都进行了评估，同时评估信号交换、免疫调节和外泌体的组织再生潜力。NTA显示外泌体粒径为100–120nm。该分析还表明，加力组和非加力组之间的外质体的形态和功能没有显著差异。. 倒置显微镜观察表明，非加力组的细胞呈纺锤形和漩涡形，随机排列。施加力后，细胞体被拉长、细长和纺锤形，每个细胞的长轴相互平行排列。随着力的增加，细胞以规则的平行方式排列。当施加至少1 g/cm2的力超过48小时时，PDLSC最终显示出漩涡状随机排列；死亡的漂浮细胞数量增加，而CCK-8活性下降。这些结果表明，弱力可以激活PDLSC分泌特定的外泌体。过度和长时间的压力会导致PDLSC萎缩和死亡。. 在施加48小时的各种力下对外泌体miRNA进行RNA测序。使用GO-term富集分析来鉴定特异性和差异性表达的miRNA。机械力诱导的PDLSC来源外泌体分泌可能反映与破骨细胞有关的炎症、免疫和成骨过程。GO和KEGG通路分析以及基于实验得出的网络图表明，大多数PDLSC外泌体中特异性和差异表达的miRNA与炎症、免疫和癌症有关。此外，许多差异性表达的miRNA尤其是miR-21与细胞成骨和破骨细胞有关。