牙髓干细胞外泌体miR-221调控巨噬细胞极化促进骨组织修复的机制研究

By the key role in transferring important biological information，exosome takes part in multiple pathophysiological processes, especially in tissue repair and regeneration. In our previous study, we applied exosomes derived from Dental Pulp Stem Cell (DPSCs) in periodontitis and they showed the ability to induce macrophage into M2 macrophage which was important in molding the local immuno-microenvironment and bone formation. To unveil its underlying mechanism, here we analyzed gene expression in macrophage and M2 macrophage obtained by plating the macrophage in the medium of DPSCs-derived-exosome, identifying miR-221 was the most significantly upregulated gene during M2 macrophage activation. RNA pull-down also showed that miR-221 can interact with Smurf1 mRNA, which was proved to down-regulate M2 macrophage activation by TGF-β pathway. Hence, we propose that exosome is involved in M2 macrophage activation through miR-221, which is capable of upregulating TGF-β pathway by inhibiting the translation of Smurf1, and finally facilitate the restoration of bone tissue. In the following step , the involvement of miR-221 in M2 macrophage activation will be determined by methods such as RISC、binding sequence knock-down, etc. Then the binding domain of miR-221 and Smurf1 will be detected, followed by several experiments to determine their relationship, so that we can verify our hypothesis that miR-221 interacting with Smurf1 triggers M2 macrophage by TGF-β pathway. This project will give us the opportunity to understand the involvement of miR-221-induced M2 macrophage in immuno-microenvironment regulation and shed new light on the therapeutic value of DPSCs in bone tissue restoration.

外泌体通过重要生物信息的传递参与组织损伤与修复等多种病理生理过程。本课题组前期研究证实牙髓干细胞外泌体可通过诱导巨噬细胞M2型极化促进骨组织损伤修复；并发现在牙髓干细胞外泌体作用下，M2型极化过程中巨噬细胞胞浆miR-221上调最为显著，且miR-221可与Smurf1转录本在胞浆结合。鉴于Smurf1是TGF-β通路的关键负调节因子，而该通路参与调控M2型极化，我们认为：外泌体miR-221可能通过抑制靶细胞Smurf1翻译、调控TGF-β通路而促进巨噬细胞M2型极化，从而参与骨组织损伤修复。为此，本研究拟采用RISC免疫共沉淀、结合序列突变等技术，分析miR-221与Smurf1结合区域及作用机理；阐明miR-221抑制Smurf1激活TGF-β通路在巨噬细胞极化中的机制，明确外泌体miR-221对巨噬细胞M2型极化的影响，为牙髓干细胞应用于骨组织损伤修复提供理论依据

外泌体通过重要生物信息的传递参与组织损伤与修复等多种病理生理过程。牙髓干细胞外泌体在损伤部位炎症免疫调控及组织再生修复过程中发挥重要作用。本项目主要研究了牙髓干细胞外泌体调控牙周免疫微环境促进骨组织修复的作用和机制，进一步探讨了病理状态下牙周组织防御体系的改变，并通过调控局部免疫微环境改善病理状态下牙周组织损伤。获得结果如下：① 为了探讨牙髓干细胞外泌体在调控巨噬细胞极化促进骨组织修复的作用，通过构建小鼠牙周炎模型，结合miRNA-seq、miRNA抑制剂及模拟物，我们发现牙髓干细胞外泌体可促进牙周炎症局部巨噬细胞向抗炎型（M2）型极化，并明确牙髓干细胞外泌体通过miR-1246调控巨噬细胞M2型极化促进小鼠牙周炎骨组织修复。② 进一步通过3D培养改性牙髓干细胞外泌体，揭示了3D培养提高牙髓干细胞外泌体miR-1246表达，并调控牙周局部免疫微环境，促进小鼠牙周炎骨组织修复。③ 为了探讨牙髓干细胞外泌体在促进牙周组织再生的作用及机制。通过体外细胞模型及大鼠牙周缺损模型，揭示了牙髓干细胞外泌体可通过调控牙周局部成骨成血管促进大鼠骨缺损修复。④ 为了进一步探究牙周组织防御体系在病理状态下的改变及机制，通过检测临床牙周组织样品及牙周上皮细胞模型，发现糖尿病状态下牙周上皮细胞发生自噬溶酶体途径破坏且内质网应激反应延长。通过转录组测序及慢病毒过表达体系，明确了ATP6V0C和SERPIN1H在病理状态下牙周组织防御体系中的作用。⑤ 最后，我们进一步探讨调控局部免疫微环境改善病理状态下牙周组织损伤，使用CCL2抑制剂bindarit治疗糖尿病牙周炎小鼠，可显著减少糖尿病牙周炎症组织中促炎型单核细胞浸润。相关结果探讨了牙髓干细胞外泌体在调控牙周免疫微环境促进骨组织修复的机制，为牙髓干细胞外泌体应用于骨组织损伤修复提供理论依据，进而为口颌面损伤修复机制的研究提供新的视角。