目的 探索牵张加载下牙周膜细胞(periodontal ligament cell,PLC)外泌体对成骨细胞分化的影响,以及牵张加载下 PLC 外泌体在力相关牙周组织改建中的调控作用。 方法 对体外培养人 PLC 施加 20% 牵张应变后,提取上清中的外泌体与成骨细胞共培养,检测成骨细胞分化情况,并探索外泌体中发挥调控作用的 miRNA。 结果 牵张加载下 PLC 分泌的外泌体可上调成骨细胞中碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素( osteocalcin,OCN)、转录因子 Osterix(Osx)、I 型胶原蛋白(type I collagen,COL-1)、Runt 相关转录因子 2(Runt-related transcription factor2, Runx2)、和骨形态发生蛋白 2(bone morphogenetic protein 2, BMP 2)等成骨相关因子表达,促进细胞成骨向分化。 牵张加载下 PLC 外泌体差异表达 miRNA 中,miRNA-181d-5p 表达量上调达 107 倍,将其转染成骨细胞后,成骨细胞中 ALP、Osx、Col-1 、Runx2、Ocn 和 BMP2 等成骨相关因子表达亦增加。 结论 本研究初步揭示了牵张加载下PLC 外泌体 miR-181d-5p 在调控成骨细胞分化中的作用,对于明确力相关牙周组织改建中的信号通路具有科学意义。

Objective To investigate the effect of exosomes, derived from periodontal ligament cells (PLCs) under stretch loading on osteoblast differentiation, and the role of these exosomes in force-related periodontal tissue remodeling. Methods After 20% strain was applied to human PLCs cultured in vitro, exosomes from the supernatant were extracted and co-cultured with osteoblasts, then the differentiation of osteoblasts was detected, and miRNA which played a regulatory role in exosomes was explored. Results Exosomes secreted by PLCs under stretch loading could up-regulate the expression of osteogenic-related factors including alkaline phosphatase(ALP), transcription factor Osterix（osterix, Osx）, type I collagen (Col-1), Runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), and bone morphogenetic protein 2 (BMP2) in osteoblasts, as well as promote osteogenic differentiation of cells. Among the miRNAs differentially expressed in PLC exosomes under stretch loading, the expression of miRNA-181d-5p was up-regulated by 107 times, and the expression of osteogenic-related factors including ALP, Osx, Col-1, Runx2, Ocn and BMP2 was also increased in osteoblasts transfected with miRNA-181d-5p. Conclusions This study preliminarily reveals the role of exosome miR-181d-5p secreted by PLCs under stretch loading in regulating the differentiation of osteoblast, which is of scientific significance for clarifying the signaling pathway in force-related periodontal tissue remodeling.

国家自然科学基金项目( 82071157,31470903,31270991,30900282),上海市卫生健康委员会卫生行业临床研究专项资助项目(201940009),上海交通大学医学院附属第九人民医院原创探索项目(JYYC002)