巨噬细胞-间充质干细胞外泌体信息交流紊乱：绝经女性牙周炎骨缺损加重的新机制

Estrogen deficiency in postmenopausal women results in the aggravation of periodontitis, which leads to progressive attachment loss and alveolar bone destruction. It is widely accepted that the imbalance of osteogenesis and osteoclastogenesis in estrogen deficiency is responsible for the observed aggravation. However, these mechanism studies usually ignored the role of inflammation under estrogen deficiency. In healthy condition, the existence of periosteum prevents the contact between the inflammatory cells in the gingiva and mesenchymal stem cells (MSCs) in the alveolar bone. Our previous study has found that in the course of periodontitis, the activation of inflammatory response leads to the destruction of periosteum, which makes the crosstalk between inflammatory cells, such as macrophages in the gingival tissue, and MSCs in the bone possible. Furthermore, exosomes, the newly identified cell-cell communication mediators, might be the big player in the crosstalk. Previous studies have found estrogen deficiency results in aberrant macrophage polarization between M1 and M2 state, causing alteration in miRNA-expression in both macrophages and exosomes they secret, which is consistent with our findings. Taken together, we here hypothesize that the imbalanced polarization of macrophages alters the expression of inflammatory miRNAs in their exosomes, which in turn endocytosed by MSCs in the alveolar bone marrow, causing inhibition of osteogenic differentiation during bone restoration in periodontitis in estrogen deficiency. To this end, the proposal here will conduct the following experiments. First, we will clarify the pathogenic role of macrophages and their exosomes in periodontitis in the context of ovariectomized mice. Then, we will explore the effects of miRNAs in macrophage derived exosomes on osteogenic differentiation of MSCs, via CRISPR/Cas9 mediated Dicer knockout in macrophages. Next, we will modify the exosomes and test their effects in vitro and in vivo. Finally, we will explore the correlation between candidate exosomal miRNAs and the severity of periodontitis in clinical samples. It would be highly possible that we will discover a potential mechanism for the aggravated periodontitis in postmenopausal women, thus shedding light on developing new strategies for the management.

绝经后女性牙周炎患者牙槽骨骨质流失更为严重。既往研究主要关注雌激素缺乏对成骨和破骨细胞自身的调控，但忽略了牙周炎炎症微环境的影响。课题组新近发现，牙周炎导致牙槽骨骨膜破坏，为牙槽骨间充质干细胞（MSCs）与牙龈炎症细胞间信息交流提供可能，而外泌体是二者交流的重要方式。此外，雌激素缺乏增强巨噬细胞M1极化，M1来源的外泌体显著抑制MSCs成骨。我们推测牙周炎合并雌激素缺乏时，牙龈中巨噬细胞更倾向于向M1极化，其分泌的外泌体跨越受损的牙槽骨骨膜后被MSCs内吞，最终抑制成骨分化和骨修复，加重牙周炎。本项目拟结合雌激素缺乏小鼠牙周炎模型、CRISPR/Cas9技术敲除巨噬细胞Dicer、临床样本等，通过外泌体干预、基因表达检测和牙周炎系统评价等手段，明确雌激素缺乏时巨噬细胞极化异常后外泌体及其关键miRNAs的变化；阐明上述改变对MSCs成骨分化和牙周炎的影响；期望发现绝经女性牙周炎加重新机制。

绝经后女性牙周炎加重。巨噬细胞异常可能是雌激素缺乏条件下的重要免疫微环境改变，间充质干细胞（MSCs）成骨功能降低是导致牙槽骨丧失的主要原因，而细胞外囊泡作为巨噬细胞和间充质干细胞的信息交流介质发挥着关键作用。本项目基于前期研究基础，探讨巨噬细胞对MSCs成骨的调控及其作用机制。主要发现包括：1）研发一种小鼠牙周炎造模方法，构建卵巢去势（OVX）及牙周炎小鼠模型，阐明雌激素缺乏可加重小鼠牙周炎；2）雌激素缺乏条件下牙周炎小鼠牙周组织巨噬细胞极化紊乱，表现为巨噬细胞M1极化增加，M2极化减弱；3）巨噬细胞来源小细胞外囊泡（sEVs）调控间充质干细胞的成骨分化，M1型巨噬细胞sEVs抑制MSCs成骨分化，M2型巨噬细胞sEVs促进其成骨分化；4）巨噬细胞sEVs中的miRNA是调控MSCs成骨分化的关键分子机制，通过干预关键miRNA可减缓OVX小鼠牙周炎发生发展。. 在此基础上，课题组还针对牙周炎免疫炎症微环境的特征，探讨了其中自然杀伤样B细胞（NKB）及其分泌的IL-18在牙周炎发生发展中的作用。此外，根据MSCs成骨分化与细胞归巢特征，设计了治疗牙周骨缺损智能新型超分子水凝胶缓释支架材料，促进牙周MSCs细胞归巢，促进牙周骨再生，为牙周炎的治疗提供了新策略。. 在该项目支持下，共发表SCI论文6篇（均已标注基金号），其中以通讯作者发表于Chem Eng J，Front Immunol，Front Bioeng Biotech和Regener Biomater，以第一作者兼共同通讯作者发表于ACS Nano。项目进行期间，申请国家发明专利2项（已授权1项）；培养毕业博士2名，毕业硕士2名，在读博士5名，在读硕士5名。