Journal of Prevention and Treatment for Stomatological Diseases ›› 2021, Vol. 29 ›› Issue (2): 81-87.DOI: 10.12016/j.issn.2096-1456.2021.02.002

• Basic Study • Previous Articles     Next Articles

Exosomes derived from lipopolysaccharide-preconditioned dental folic cells regulate osteogenic differentiation of periodontal ligament cell in periodontitis

SHI Weiwei1,2,3(),DING Yi3,TIAN Weidong1,2,4,GUO Shujuan1,2,3()   

  1. 1. Engineering Research Center of Oral Translational Medicine, Ministry of Education, Sichuan University, Chengdu 610041, China
    2. National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
    3. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Department of Periodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
    4. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
  • Received:2020-02-19 Revised:2020-04-28 Online:2021-02-20 Published:2020-12-21
  • Contact: Shujuan GUO
  • Supported by:
    grants from National Key Research and Development Program of China(2017YFA0104800)

脂多糖预处理的牙囊细胞外泌体对牙周炎牙周膜细胞成骨分化的影响

石维薇1,2,3(),丁一3,田卫东1,2,4,郭淑娟1,2,3()   

  1. 1.口腔转化医学教育部工程研究中心 四川大学,四川 成都(610041)
    2.口腔再生医学国家地方联合工程实验室 四川大学华西口腔医院,四川 成都(610041)
    3.口腔疾病研究国家重点实验室 国家口腔疾病临床医学研究中心 四川大学华西口腔医院牙周病科,四川 成都(610041)
    4.口腔疾病研究国家重点实验室 国家口腔疾病临床医学研究中心 四川大学华西口腔医学院创伤与整形外科,四川 成都(610041)
  • 通讯作者: 郭淑娟
  • 作者简介:石维薇,硕士,Email: 494808794@qq.com
  • 基金资助:
    国家重点研究开发项目(2017YFA0104800)

Abstract:

Objective The purpose of this study was to investigate the effect of different concentrations of exosomes (Exos) secreted from dental folic cells (DFCs) preconditioned with lipopolysaccharide (LPS) on the osteogenic differentiation ability of periodontal cells in periodontitis (p-PDLCs) in patients to provide a basis for the prevention and treatment of periodontal disease. Method Tissue block and enzyme digestion methods were used to culture DFCs and p-PDLCs. Exosomes were isolated from 250 ng/mL LPS-preconditioned DFCs 24 h later. The characteristics of exosomes were detected by transmission electron microscopy, particle size analysis and Western blotting. The effects of 10 μg/mL and 100 μg/mL exosomes on the osteogenic differentiation of p-PDLCs were detected by RT-PCR and Alizarin red staining. Results LPS-pretreated DFC-derived exosomes (L-Exos) are vesicle-like structures with a size between 30-100 nm that positively express CD63 and Alix. Compared with the control group, exosomes significantly upregulated Periostin, Col Ⅰ, and Col Ⅲ expression at 100 μg/mL (P < 0.05), while TGF- β1 was significantly upregulated at 10 μg/mL (P < 0.01). At 7 days after osteogenic induction, mineralized nodules were significantly more abundant in the exosome group than in the control group (P < 0.01), and the results were better at a concentration of 100 μg/mL (P < 0.01). Conclusion 100 μg/mL L-Exos are better than 10 μg/mL L-Exos in enhancing the osteogenic differentiation ability of p-PDLCs.

Key words: lipopolysaccharide, dental folic cells, periodontal ligament cells, exosomes, osteogenic differentiation, collagen fiber, mineralization, periodontal regeneration

摘要:

目的 探究脂多糖(lipopolysaccharide,LPS)预处理牙囊细胞(dental folic cells,DFCs)外泌体(exosomes,Exos)在不同浓度条件下对牙周炎患者的牙周膜细胞(periodontal ligament cells of periodontitis, p-PDLCs)成骨分化能力的影响,为牙周病的防治提供基础。方法 组织块酶消化法培养DFCs及p-PDLCs,提取经250 ng/mL LPS刺激DFCs 24 h后的Exos,Exos的表征通过透射电镜扫描、粒径分析以及蛋白印记法进行鉴定;通过逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)、茜素红染色等方法检测10 μg/mL以及100 μg/mL浓度的Exos对p-PDLCs成骨向分化能力的影响。结果 LPS预处理的DFCs外泌体(L-Exos)为直径30~100 nm之间的囊泡样结构,高表达CD63和相互作用蛋白X(ALG-2 interacting protein-X,Alix)。100 μg/mL L-Exos上调p-PDLCs骨膜蛋白、Ⅰ型胶原、Ⅲ型胶原基因的表达(P < 0.05);而10 μg/mL L-Exos仅上调p-PDLCs的转化生长因子-β1(transforming growth factor-β1, TGF-β1)基因的表达(P < 0.01)。在成骨诱导7 d后,L-Exos组的矿化结节明显多于对照组(P < 0.01),并且100 μg/mL L-Exos组的矿化效果更佳(P < 0.01)。结论 100 μg/mL L-Exos较10 μg/mL L-Exos更有利于增强p-PDLCs成骨向分化能力。

关键词: 脂多糖, 牙囊细胞, 牙周膜细胞, 外泌体, 成骨分化, 胶原纤维, 矿化, 牙周再生

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