Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Mar 20;43(3):420-427.
doi: 10.12122/j.issn.1673-4254.2023.03.12.

[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]

[Article in Chinese]
Y Liu  1 , L Zeng  2 , W Wang  1 , Y Yang  2 , Z Wang  2 , J Liu  2 , W Li  2 , J Sun  2 , X Yu  2
Affiliations

[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]

[Article in Chinese]
Y Liu et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

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.

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。

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

PubMed Disclaimer

Figures

图 1
图 1
hBMMSCs中外泌体鉴定 Identification of exosomes from human bone marrow mesenchymal stem cells (hBMMSCs). A: Transmission electron microscopy of the exosomes. B-D: Western blotting for detecting the expressions of CD9 and CD81 in the exosomes. *P < 0.05.
图 2
图 2
hBMMSCs外泌体摄取实验 Exosome uptake assay of hBMMSCs. A: PKH67 labeling (green) of the exosomes and DAPI staining (blue) of the nuclei. B: Changes of average fluorescence intensity overtime in human periodontal ligament stem cells (hPDLSCs) co-cultured with the exosomes. C: qRT-PCR for detecting miR-335-5p expression. **P < 0.01.
图 3
图 3
hBMMSCs外泌体促进成骨分化 Exosomes derived from hBMMSCs promote osteogenic differentiation of hPDLSCs in vitro. A-F: qRT-PCR detection of mRNA expressions of IL-1β (A), IL-6 (B), IL-8 (C), BMP-2 (D), OCN (E), and RunX2 (F). G: Alizarin red staining of hPDLSC cultures (×200). H: ALP staining of hPDLSC cultures (Original magnification: ×200). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
图 4
图 4
hBMMSCs-exo来源的miR-335-5p调控hPDLSCs成骨分化 MiR-335-5p derived from hBMMSCs exosomes regulates osteogenic differentiation of hPDLSCs. A-G: qRT-PCR detected expression of miR-335-5p (A) and mRNA levels of IL-1β (B), IL-6 (C), IL-8 (D), BMP-2 (E), OCN (F), RUNX2 (G). H: Alizarin red staining of hPDLSC cultures (×200). I: ALP staining of hPDLSC cultures (×200). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
图 5
图 5
hBMMSCs外泌体来源的miR-335-5p通过DKK1促进hPDLSCs成骨分化 MiR-335-5p from hBMMSC exosomes promote osteogenic differentiation of hPDLSCs via DKK1. A: Starbase database analysis for predicting the binding sequences between miR-335-5p and DKK1. B: Dual-luciferase reporter gene assay for verifying the targeting relationship between miR-335-5p and DKK1. C, D: Western blotting for detecting DKK1 protein expression in hPDLSCs. E-J: qRT-PCR for detecting mRNA levels of IL-1β (E), IL-6 (F), IL-8 (G), BMP-2 (H), OCN (I), and RunX2 (J). K: Alizarin red staining of hPDLSC cultures (×200). L: ALP staining of hPDLSC cultures (×200). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Sanz M, Ceriello A, Buysschaert M, et al. Scientific evidence on the links between periodontal diseases and diabetes: consensus report and guidelines of the joint workshop on periodontal diseases and diabetes by the International diabetes Federation and the European Federation of Periodontology. Diabetes Res Clin Pract. 2018;137:231–41. doi: 10.1016/j.diabres.2017.12.001. - DOI - PubMed
    1. Kinane DF, Stathopoulou PG, Papapanou PN. Periodontal diseases. Nat Rev Dis Primers. 2017;3:17038–45. doi: 10.1038/nrdp.2017.38. - DOI - PubMed
    1. Rajendran M, Looney S, Singh N, et al. Systemic antibiotic therapy reduces circulating inflammatory dendritic cells and treg-Th17 plasticity in periodontitis. J Immunol. 2019;202(9):2690–9. doi: 10.4049/jimmunol.1900046. - DOI - PMC - PubMed
    1. Preus HR, Gjermo P, Baelum V. A randomized double-masked clinical trial comparing four periodontitis treatment strategies: 5-year tooth loss results. J Periodontol. 2017;88(2):144–52. doi: 10.1902/jop.2016.160332. - DOI - PubMed
    1. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004;364(9429):149–55. doi: 10.1016/S0140-6736(04)16627-0. - DOI - PubMed

Publication types

LinkOut - more resources