牙周炎症微环境中牙周膜干细胞来源的外泌体对巨噬细胞功能的影响

The unclear mechanism of periodontitis has restrained the clinical therapeutic strategies. The macrophage function is a hub for inflammation regulation and bacteria can directly influence its polarization direction, which is considered as the initial factor of periodontitis. However, more and more researches indicate that the polarization of macrophages is a complicated regulation network and whether there is an indirect regulation pathway between bacteria and macrophages is unclear. The mesenchymal stem cells (MSC) is able to regulate various cell types through different ways, among which the exosomes have participated in the regulation loop. In particular, during inflammatory diseases pathology, the exosomes play important roles for macrophage polarization regulation. Therefore, we infer that in the periodontitis microenvironment, the PDLSC-derived exosomes may be involved in the indirect regulation between bacteria and macrophages. To test this hypothesis, the LPS is used to simulate periodontitis microenvironment in our current project and the potential influence of PDLSC-derived exosomes on macrophage polarization is analyzed in detail by using molecular biological methods such as gene sequencing and western blot. In addition, the molecular mechanism of PDLSC-derived exosomes alteration caused by inflammation is also investigated. Our study may provide a new insight into the mechanisms of periodontitis, and may also help to find new target for periodontitis therapy.

牙周炎发病机理不明是制约临床治疗效果的关键，巨噬细胞功能表现是调控炎症反应的重要枢纽。细菌等因素能直接影响巨噬细胞极化方向，是牙周炎的始动因素。但巨噬细胞极化的调控是一个复杂的网络，是否存在其他的间接调控通路目前尚不明确。间充质干细胞MSC由于对多种细胞具有调控潜能而备受重视，其中外泌体作为新近发现的细胞间交流重要介质，参与了MSC 对其他细胞的调控作用，特别是在炎症相关疾病的发生发展中对巨噬细胞极化具有重要调控作用。因此我们推测在牙周炎症微环境中，牙周膜干细胞PDLSC来源的外泌体可能参与了对巨噬细胞极化的间接调控作用。为验证这一假说，本课题拟采用LPS刺激模拟牙周炎症微环境，采用基因测序、蛋白印迹等分子生物学手段对PDLSC分泌的外泌体对巨噬细胞极化的可能影响和机制进行深入分析，并阐述炎症影响PDLSC外泌体变化的分子机制，为牙周炎发病机理提供新思路，并开发新的牙周炎治疗靶点。

牙周炎的组织破坏与机体过度宿主免疫反应相关；如何调控免疫细胞功能从而抑制炎症进展是完善牙周炎治疗体系的重要环节。固有免疫系统的巨噬细胞以其M1、M2表型在炎症进展中发挥枢纽作用。牙周膜干细胞（PDLSCs）是牙周免疫调控的主要功能细胞来源，但正常及炎症状态下PDLSCs的功能存在巨大差异，尤其是炎症微环境状态下PDLSC如何调控巨噬细胞极化尚无明确报道。本课题以外泌体为切入点，进一步研究炎症微环境中PDLSCs对巨噬细胞功能的影响及可能的分子调控机制，对牙周炎发病机理进行补充完善，为牙周炎治疗提供候选靶点及理论依据。首先从健康或牙周炎不同来源牙周组织中分离提取PDLSCs并鉴定；同时以LPS刺激健康PDLSCs模拟炎症微环境，分别与人白血病细胞系（THP-1）来源的巨噬细胞通过Transwell体系进行间接共培养，发现炎症环境下PDLSCs在mRNA和蛋白质水平均可下调巨噬细胞的M2极化；差速离心法提取健康及炎症环境下PDLSCs来源外泌体并作用于巨噬细胞，结果显示炎症环境下PDLSCs来源外泌体可促进巨噬细胞M1极化；抑制其外泌体分泌后，巨噬细胞M1极化效应减弱；miRNA芯片结果显示miR-143-3p在炎症环境下PDLSCs来源外泌体中的表达较健康来源显著上调，双荧光素酶报告基因实验结果显示miR-143-3p可靶向调控PI3Kγ的表达；向巨噬细胞内进行miR-143-3p转染，发现其可促进巨噬细胞M1极化上调，同时参与了PI3K/AKT/NF-κB信号通路的调控；而使用PI3K/AKT信号通路激活剂可逆转以上效应；随后又以转染miR-143-3p后PDLSCs来源外泌体处理巨噬细胞，也观察到同样现象，提示外泌体可携带miR-143-3p通过调控PI3K/AKT/NF-κB信号通路上调巨噬细胞M1型极化。此外，还发现炎症环境下PDLSCs内质网应激（ERs）水平明显高于健康PDLSCs，且PERK通路参与了该ERs信号进一步向巨噬细胞的传递，调控其向M1方向极化，但干细胞ERs与外泌体分泌之间的关系尚未明确。本研究的科学意义在于：明确炎症微环境中PDLSCs 的ERs水平明显升高，其可通过外泌体转运miR-143-3p调控巨噬细胞PI3K/AKT/NF-κB信号通路从而上调巨噬细胞M1极化，亦可通过PERK通路相关的ERs信号传递上调巨噬细胞M1极化。