牙周膜干细胞来源的外泌体在牙周组织再生中的作用及机制研究

Periodontitis is a common oral disease with the destruction of periodontal tissues, including alveolar bone, periodontal ligament and cementum, which leads to the loosening and loss of teeth. With the wide application of tissue engineering, the investigation of stem cells in the regeneration of periodontal complex tissue has attracted more attentions. It was reported that periodontal ligament stem cells (PDLSCs) were applied for periodontal regeneration due to their proliferation and multiple differentiation potential. Despite such success in preclinical models, little is known about how the transplanted PDLSCs can actually contribute to regeneration. Recently, accumulating evidence has shown that exosomes, the naturally secreted nanocarriers of cells, play crucial roles in cell-based tissue regeneration, because they are primary biological mediators of intercellular communication by transferring proteins and genetic information to target cells. In the current study, we will investigate the cell interaction by in vitro indirect co-culture systems to elucidate the cross talk between PDLSCs and bone marrow mesenchymal stem cells (BMMSCs), by soluble mediators only. Then exosomes derived from PDLSCs (PDLSC-exo) are isolated and characterized via electron microscopy and microRNA expression profiling. The effects of PDLSC-exo on the behavior of BMMSCs in terms of cell proliferation, migration and osteogenic differentiation in vivo will be evaluated. In an in vivo rat maxillary periodontal defect model, the contribution of transplanted PDLSC-exo are assessed by histological examination.

牙周组织疾病是发生在牙周膜、牙槽骨和牙骨质等支持组织的常见疾病，会引起牙齿松动甚至缺失。牙周膜干细胞是牙周再生的重要种子细胞，在动物实验等前期研究中获得了良好的牙周再生效果，但其参与牙周组织再生的机制尚不明确。外泌体作为细胞旁分泌的产物，通过在细胞之间传递蛋白和基因等信号因子，进行胞间信息交流。近年来研究表明，种子细胞移植后通过外泌体募集宿主细胞参与组织再生。本课题建立间接共培养模型，模拟牙周膜干细胞与骨髓间充质干细胞的关系，明确牙周膜干细胞是否能够通过外泌体促进骨髓间充质干细胞成骨分化。然后提取牙周膜干细胞来源的外泌体，用透射电镜观察其形态，用基因芯片检测其microRNA构成。将牙周膜干细胞来源的外泌体直接作用于骨髓间充质干细胞，分析其对骨髓间充质干细胞增殖、迁移和成骨分化能力的影响。最后，用牙周膜干细胞来源的外泌体作用于牙周缺损动物模型，采用形态学和组织化学手段评估牙周组织再生效果。

牙周组织疾病是发生在牙周膜、牙槽骨和牙骨质等支持组织的常见疾病，会引起牙齿松动甚至缺失。牙周膜干细胞是牙周再生的重要种子细胞，在动物实验等前期研究中获得了良好的 牙周再生效果，但其参与牙周组织再生的机制尚不明确。外泌体作为细胞旁分泌的产物，通过在细胞之间传递蛋白和基因等信号因子，进行胞间信息交流。近年来研究表明，种子细胞移植后通过外泌体募集宿主细胞参与组织再生。.本课题建立间接共培养模型，模拟牙周膜干细胞与骨髓间充质干细胞的关系，证明牙周膜干细胞能够通过旁分泌路径促进骨髓间充质干细胞成骨分化，敲降Rab 27a后牙周膜干细胞的促成骨能力下降。采用经典的超速离心法，提取牙周膜干细胞来源的外泌体，通过形态学观察和表面标志物进行鉴定。将牙周膜干细胞来源的外泌体直接作用于骨髓间充质干细胞，表明其能够促进骨髓间充质干细胞增殖、迁移和成骨分化能力。RNA-seq和功能实验提示，牙周膜干细胞来源的外泌体通过腺苷受体通路促进靶细胞Akt/Erk磷酸化。将牙周膜干细胞来源的外泌体局部应用于大鼠颅骨极限缺损模型，采用形态学和组织化学手段评估显示，外泌体能够促进缺损区的细胞增殖和骨再生修复。.本课题结果表明，牙周膜干细胞来源的外泌体能够促进细胞迁移和成骨分化从而促进骨组织再生，有望应用于非细胞疗法的牙周组织工程骨组织工程。.