外泌体对脂多糖诱导的牙髓干细胞成骨分化的作用及代谢机制

口腔生物医学 ›› 2021, Vol. 12 ›› Issue (4): 219-224.

外泌体对脂多糖诱导的牙髓干细胞成骨分化的作用及代谢机制

郭佳¹,张哲儒²,雷啸¹,金岩³,李蓓⁴,金钫¹

1. 空军军医大学第三附属医院口腔正畸科
2. 中国人民解放军联勤保障部队第九二五医院五官科
3. 空军军医大学第三附属医院口腔组织病理学教研室
4. 空军军医大学第三附属医院组织工程中心

收稿日期:2021-10-13 修回日期:2021-12-08 出版日期:2021-12-25 发布日期:2022-01-04
通讯作者: 金钫 E-mail:fangjin@fmmu.edu.cn

Effects of exosomes on the osteogenic differentiation of dental pulp stem cells induced by lipopolysaccharide and its metabolic mechanism

Received:2021-10-13 Revised:2021-12-08 Online:2021-12-25 Published:2022-01-04

摘要/Abstract

摘要： 目的：探究外泌体对脂多糖（LPS）诱导的牙髓干细胞（DPSCs）成骨分化的影响。方法：收集健康成人和儿童的脱落乳牙牙髓干细胞（SHED），分离、培养DPSCs和SHED并用流式细胞仪鉴定。收集SHED上清的外泌体，使用透射电镜、Western blot和粒径分析进行鉴定；检测对照组（10 mg/L PBS）、LPS组（10 mg/L LPS）和LPS+外泌体组（10 mg/L LPS+10 mg/L 外泌体）的DPSCs细胞外酸化率（ECAR）和耗氧率（OCR）水平；加入线粒体呼吸酶抑制剂抗霉素A后，实时定量RT-PCR和茜素红染色检测各组DPSCs的成骨分化能力。结果：成功获得DPSCs和SHED来源外泌体；LPS可以提高DPSCs的ECAR水平，使OCR/ECAR水平下降，而加入外泌体后，DPSCs的OCR水平升高，OCR/ECAR水平得到恢复；外泌体可以恢复LPS诱导的成骨分化下降，而加入抗霉素A可以抑制外泌体的促成骨分化能力。结论：SHED来源的外泌体通过提高OCR/ECAR水平，来促进炎症微环境下DPSCs的成骨分化能力。

关键词: 外泌体, 牙髓干细胞, 炎症微环境, 成骨分化, 代谢

Abstract: Objective:To explore the effect of exosomes on the osteogenic differentiation of dental pulp stem cells (DPSCs) induced by lipopolysaccharide (LPS). Methods:Stem cells from human exfoliated deciduous teeth (SHED) from deciduous teeth of healthy adults and children were collected. DPSCs and SHED were isolated, cultured and identified by flow cytometry. Exosomes were collected from the supernatant of SHED and identified by transmission electron microscopy, Western blot and nanoparticle tracking analysis. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) levels of the control group (10 mg/L PBS), LPS group (10 mg/L LPS) and LPS + exosomes group (10 mg/L LPS+10 mg/L exosomes) were examined; The osteogenic differentiation ability of each group was detected by real-time fluorescent quantitative PCR (RT-PCR) and Alizarin Red staining after addition of mitochondrial respiratory enzyme inhibitor AA. Results: DPSCs and exosomes derived from SHED were successfully obtained. LPS could increase the ECAR level of DPSCs and decrease the OCR/ECAR ratio. After adding exosomes, the level of OCR increased and the decreased OCR/ECAR ratio was restored; RT-PCR and Alizarin Red staining results showed that exosomes can restore the decreased osteogenic differentiation induced by LPS, and AA can inhibit the enhanced osteogenic differentiation ability of exosomes. Conclusions: SHED-derived exosomes can increase the OCR/ECAR ratio of DPSCs to promote osteogenic differentiation in the inflammatory microenvironment.

Key words: exosome, ?dental pulp stem cells, ?inflammatory microenvironment, ?osteogenesis differentiation, ?metabolism

郭佳张哲儒雷啸金岩李蓓金钫. 外泌体对脂多糖诱导的牙髓干细胞成骨分化的作用及代谢机制[J]. 口腔生物医学, 2021, 12(4): 219-224.

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