牙囊干细胞来源外泌体调控牙周膜干细胞促进牙周组织再生的相关研究

Periodontitis is a common clinical disease but hard to solve. To study the regeneration mechanism of periodontal tissue has important scientific significance. Tissue engineering-based cell transplantation is the main strategy for periodontal tissue regeneration. It is increasing apparent that MSCs stimulate the activity of tissue-resident recipient cells via paracrine mechanisms to tissue repair. Exosomes, the naturally secreted nanocarriers from cells, play key roles in intercellular communication, tissue homeostasis and regeneration. In our previous studies, periodontium has been reconstructed by using dental follicle stem cells (DFSCs) sheet, but the mechanism is unknown. In this study, exosomes from DFSCs will be chosen to find their function on prolife, migration, osteogenesis of periodontal ligament stem cells. These exosomes will be transplanted into model animals by cell-free method to study the periodontal regeneration. The unique molecular (miRNA or protein) in exosomes from DFSCs will be screened by using Next-Generation Sequencing and iTRAQ, the molecular structure and fuction will be predicted by bioinformatics analysis and confirmed by gene transfection or miRNA intervention. In addition, regulatory mechanism of signal molecular in Cell transplantation periodontal tissue regeneration will be studied.

牙周炎是临床常见而亟待解决的难题。基于细胞移植的组织工程是目前再生牙周组织的主要策略，研究其再生机制具有重要的科学意义。Exosomes作为旁分泌的重要组成已被证实在组织再生中扮演关键角色。课题组前期研究利用牙囊干细胞膜片成功再建了牙周组织样结构，但牙囊干细胞在牙周再生中的作用仍不清楚。本项目拟以牙囊干细胞exosomes为研究重点，确定其对牙周膜干细胞增殖、迁移、成骨分化等的作用，在动物模型中以cell-free的植入方式进行牙周组织再生的研究，为建立不依赖细胞的牙周组织工程技术奠定实验基础。同时，采用miRNA高通量测序和iTRAQ技术，分别从miRNA和分泌蛋白水平，鉴定牙囊干细胞exosome特有组分，并运用生物信息学策略初步预测其结构和功能，通过基因转染、miRNA干扰等技术进行验证，从而进一步探讨细胞移植再生牙周组织的信号分子调控机制。

背景：牙周炎是临床常见而亟待解决的难题。基于细胞移植的组织工程是目前再生牙周组织的主要策略，研究其再生机制具有重要的科学意义。外泌体（exosomes）作为旁分泌的重要组成已被证实在组织再生中扮演关键角色。课题组前期研究利用牙囊干细胞（Dental follicle stem cells，DFSCs）膜片成功再建了牙周组织样结构，但牙囊干细胞在牙周再生中的作用仍不清楚。本项目拟以牙囊干细胞来源外泌体（exosomes derived from DFSCs，DFSCs-exosomes）为研究对象，通过体外实验探究DFSCs-exosomes对牙周膜干细胞（periodontal ligament stem cells，PDLSCs）增殖、迁移、成骨分化等生物学特性的影响，通过体内实验验证DFSCs-exosomes再生牙周组织的可能，从而为探索无细胞移植技术再生牙周组织提供理论基础。.方法：分离并鉴定DFSCs-exosomes，将DFSCs-exosomes与PDLSCs共培养；通过EdU、CCK-8、细胞周期实验、划痕实验、茜素红染色、Q-PCR及western blot检测DFSCs-exosomes对PDLSCs生物学特性的影响；通过转录组测序及功能富集分析寻找可能被DFSCs-exosomes激活的信号通路并进一步验证；将负载有DFSCs-exosomes的到胶原膜移植到SD大鼠的牙周缺损部位，通过HE染色和micro-CT评估牙周组织再生情况。.结果： DFSCs-exosomes被PDLSCs摄取后促进了PDLSCs增殖及迁移；DFSCs-exosomes促进PDLSCs矿化结节形成，并上调成骨相关基因（RUNX2, BSP, COL1）和成骨相关蛋白（RUNX2, BSP, COL1, ALP）的表达；DFSCs-exosomes通过激活p38 MAPK信号通路促进PDLSCs增殖；移植DFSCs-exosomes后，大鼠牙周缺损区见新牙周膜样结构和新骨形成，在新形成的牙周膜和缺损区软组织中观察到PKH26标记的DFSCs-exosomes。.结论：本研究表明DFSCs-exosomes通过促进PDLSCs增殖、迁移及成骨向分化从而促进牙周组织再生。DFSCs-exosomes促进PDLSCs增殖的作用可能部分归因于p38 MAPK信号通路的激活。