巨噬细胞外泌体对牙周膜干细胞再生潜能的调控及其机制研究

Interplay between macrophages (Mφs) and periodontal ligament stem cells (PDLSCs) at the periodontal defacts plays cricial roles for new tissue periodontal formation. Although our previous data have showed that polarized Mφs (e.g. M2 phenotypes) can yield anti-inflammatory and immunomodulatory cytokines positively affecting mesenchymal stem cells (MSCs), the underlying mechanisms remain largely unexplored, leading to a lack of effective strategies that can be used to modulate the reparative cell properties and thereafter the regenerative process. In this project, exosomes (EXs) secreted by polarized Mφs (Mφ-EXs) will be isolated from cell culture medium using a current Good Manufacturing Practice-compliant tangential flow filtration system and characterized. The classes of proteins, cytokines and RNAs present in Mφ-EXs, including coding RNAs (messenger RNAs) and noncoding RNAs (microRNAs, circular RNAs and long noncoding RNAs) will be screened and selected based on their positive influences on co-cultured PDLSCs. The bioactivity and function of the selected Mφ-EXs will be evaluated using both single cell and co-culture cell models. In this way, mechanisms underlying the role of Mφ-EXs in the regulation of PDLSCs will be uncovered. Finally, ectopic transplantation and in situ models will be established and optimized Mφ-EXs will be used to investigate how the modulation of interplay between Mφs and PDLSCs will contribute periodontal tissue regeneration. Finding from this project will provide new insight into the wound healing cascade and inform the design and development of periodontal tissue engineering strategies for tissue regeneration. Besides, our data pave the way of biological studies on the interrogation of cell-cell interplays in many other field, and facilitate the used of stem cells to cure disease and to regrow new tissue for benefit of our patients.

巨噬细胞（Mφs）和牙周膜干细胞（PDLSCs）在牙周缺损区域相互作用对再生结果产生重要影响，但机理不清，因此针对性调控策略难以建立。本项目拟探索组织再生过程中，Mφ外泌体（Mφ-EXs）对PDLSCs的调控作用和机理。梯度过滤法获取和鉴定不同极化转态下Mφ-EXs，分别筛选其参与再生正向调控的关键蛋白、分子和RNA，鉴定其结构与功能；通过体外共培养模型，系统评价Mφ-EX对PDLSCs的调控作用，深入剖析其机理，从而筛选牙周再生过程中Mφ-Exs的关键MicroRNA谱。最后通过异位、原位组织再生模型，观察Mφ-EXs调控PDLSCs和促进牙周再生的效果。本项目研究立足揭示Mφs调控牙周再生机理，不仅为牙周组织工程设计中，正向调控再生细胞功能，促进组织再生提供依据；同时，为进一步阐释多种生物学活动中，细胞-细胞相互影响提供新思路，为细胞移植用于疾病治疗开辟新的研究空间。

基于巨噬细胞（Mφs）和牙周膜干细胞（PDLSCs）在牙周缺损区域相互作用对组织再生产生的重要影响，本项目对牙周组织再生过程中，Mφ外泌体（Mφ-EXs）对PDLSCs的调控作用和机理进行了深入探索和分析。首先梯度过滤法获取不同极化状态下Mφ-Exs并鉴定其结构与功能，系统评价了不同类型Mφ-EXs对PDLSCs成骨、成软骨和成牙骨质分化的作用，筛选出了促进牙周组织再生的M2型巨噬细胞来源外泌体；进一步，筛选外泌体参与再生正向调控的关键分子并明确其促进组织再生的信号通路；最后通过异位、原位组织再生模型，在动物体内证实了Mφ-EXs调控PDLSCs和促进牙周组织再生的效果。本项目的研究结果不仅为牙周组织工程设计中，正向调控再生细胞功能、促进组织再生提供依据；同时，为进一步阐释多种生物学活动中，细胞-细胞相互影响提供新思路，为细胞移植用于疾病治疗开辟新的研究空间。本项目共发表基金标注SCI论文9篇，参与获得陕西省科技进步一等奖1项(第8参与人)，在此研究基础上，新获批了2022年国家自然科学基金面上项目，此外项目负责人担任陕西省口腔医学会牙周专委会青年委员，参编国内专著1部，培养研究生5人。