南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (3): 420-427.doi: 10.12122/j.issn.1673-4254.2023.03.12

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人骨髓间充质干细胞外泌体来源的miR-335-5p促进人牙周膜干细胞的成骨分化：基于下调DKK1表达

刘 屿，曾 莲，王卫红，杨艳玲，王 洲，刘建启，李 卫，孙婧宇，余晓宏   

  1. 昆明医科大学附属口腔医院口腔颌面外科，云南 昆明 650106；云南大学附属医院（云南省第二人民医院、云南省眼科医院）口腔内科，云南 昆明 650021
  • 出版日期:2023-03-20 发布日期:2023-03-20

Human bone marrow mesenchymal stem cell exosome-derived miR-335-5p promotes osteogenic differentiation of human periodontal ligament stem cells to alleviate periodontitis by downregulating DKK1

LIU Yu, ZENG Lian, WANG Weihong, YANG Yanling, WANG Zhou, LIU Jianqi, LI Wei, SUN Jingyu, YU Xiaohong   

  1. Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital of Kunming Medical University, Kunming 650106, China; Department of Stomatology, Affiliated Hospital of Yunnan University (Second People's Hospital of Yunnan Province, Yunnan Province Ophthalmology Hospital), Kunming 650021, China
  • Online:2023-03-20 Published:2023-03-20

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摘要： 目的 探讨人骨髓间充质干细胞（hBMMSCs）外泌体来源的 miR-335-5p 调控 DKK1 对人牙周炎中牙周膜干细胞（PDLSCs）成骨分化的影响及其作用机制。方法 提取hBMMSCs外泌体，通过透射电镜、Western blot以及PKH67标记鉴定外泌体，通过TNF-α诱导PDLSCs构建牙周炎细胞模型。提取的外泌体与TNF-α诱导的PDLSCs共同培养。qRT-PCR检测miR-335-5p，促炎因子 IL-1β、IL-6、IL-8 和成骨标志基因 RunX2、OCN、BMP-2 mRNA 表达。茜素红和 ALP 染色检测钙结节，Western blot检测DKK1蛋白表达，双荧光素酶报告实验验证miR-335-5p与DKK1的靶向关系。结果 提取的hBMMSCs外泌体中CD9和CD81显著表达（P<0.05）。hBMMSC外泌体降低TNF-α诱导的hPDLSCs中促炎细胞因子IL-1β（P<0.01）、IL-6（P<0.05）、IL-8（P<0.05）的mRNA表达并促进成骨标志基因RunX2（P<0.01）、OCN（P<0.05）、BMP-2（P<0.001）mRNA和钙结节生成。miR-335-5p在hBMMSCs外泌体中高表达，过表达miR-335-5p靶向下调DKK1（P<0.001），抑制促炎细胞因子IL-1β、IL-6、IL-8的表达（P<0.001），促进成骨标志物BMP-2、OCN、RunX2的 mRNA表达以及钙结节生成（P<0.001）。结论 hBMMSC外泌体来源的miR-335-5p靶向下调DKK1，促进hPDLSCs成骨分化，抑制牙周炎的发展进程。

关键词: 牙周炎；骨髓间充质干细胞；外泌体；miR-335-5p；牙周膜干细胞；成骨分化

Abstract: Objective To observe the effect of miR-335-5p derived from human bone marrow mesenchymal stem cell (hBMMSCs) exosomes on osteogenic differentiation of human periodontal ligament stem cell (PDLSCs) model of periodontitis and explore its mechanism. Methods The exosomes extracted from hBMMSCs were identified by transmission electron microscopy, Western blotting and PKH67 labeling. The human PDLSC model of TNF-α-induced periodontitis were co-cultured with the extracted exosomes, and qRT-PCR was performed to detect the changes in the expressions of miR-335-5p and the mRNA levels of pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) and the osteogenic marker genes (RunX2, OCN and BMP-2). Alizarin red staining and ALP staining were used to detect the formation of calcium nodules in the treated cells, and the expression level of DKK1 protein was detected with Western blotting. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-335-5p and DKK1. Results High expressions of CD9 and CD81 were detected in the extracted hBMMSC exosomes (P<0.05). In TNF-α-induced hPDLSCs, treatment with the extracted exosomes significantly reduced the mRNA expressions of IL-1β, IL-6 and IL-8, enhanced the mRNA expressions of RunX2, OCN, and BMP-2, and promoted the formation of calcium nodules. MiR-335-5p was highly expressed in hBMMSC-derived exosomes, and overexpression of miR-335-5p significantly downregulated DKK1 protein expression, inhibited the mRNA expressions of IL-1β, IL-6 and IL-8, and promoted the mRNA expressions of osteogenic markers and the formation of calcium nodules in hPDLSCs. Conclusion HBMMSC exosome-derived miR-335-5p promotes osteogenic differentiation of hPDLSCs and inhibits the development of periodontitis by downregulating DKK1.

Key words: periodontitis; bone marrow mesenchymal stem cells; exosomes; miR-335-5p; periodontal ligament stem cells; osteogenic differentiation