中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (25): 3961-3967.doi: 10.12307/2022.399

• 干细胞外泌体 Stem cell exosomes • 上一篇    下一篇

缺氧处理牙髓干细胞外泌体诱导M2巨噬细胞极化

谭  旭1，梁  羽2，梁  燕1，廖  健1   

  1. 1贵州医科大学口腔医学院/附属口腔医院，贵州省贵阳市   550004；2贵州省人民医院口腔科，贵州省贵阳市   550002
  • 收稿日期:2019-12-24 接受日期:2020-03-03 出版日期:2022-09-08 发布日期:2022-01-25
  • 通讯作者: 廖健，博士，教授，主任医师，博士生导师，贵州医科大学口腔医学院/附属口腔医院，贵州省贵阳市 550004
  • 作者简介:谭旭，女，1988年生，汉族，2014年北京大学毕业，博士，副主任医师，主要从事牙髓干细胞工程研究。
  • 基金资助:
    国家自然科学基金(82060207)，项目负责人：廖健；贵州省教育厅青年科技人才成长项目(黔教合KY字[2018]195号)，项目负责人：谭旭

Hypoxia-treated dental pulp stem cell exosomes induce M2 macrophage polarization

Tan Xu1, Liang Yu2, Liang Yan1, Liao Jian1   

  1. 1Medical School of Stomatology/Affiliated Stomatological Hospital, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 2Department of Stomatology, Guizhou Provincial People’s Hospital, Guiyang 550002, Guizhou Province, China
  • Received:2019-12-24 Accepted:2020-03-03 Online:2022-09-08 Published:2022-01-25
  • Contact: Liao Jian, MD, Professor, Chief physician, Doctoral supervisor, Medical School of Stomatology/Affiliated Stomatological Hospital, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • About author:Tan Xu, MD, Associate chief physician, Medical School of Stomatology/Affiliated Stomatological Hospital, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Supported by:
    the National Natural Science Foundation of China, Grant No. 82060207 (to LJ); Youth Science and Technology Talents Growth Project of Guizhou Education Department, Grant No. Qian Jiao He KY Zi[2018]195 (to TX)

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摘要：

文题释义：
牙髓干细胞外泌体：外泌体是由“内吞-融合-外排”等一系列调节过程形成的双层类脂囊泡。牙髓干细胞是存在于牙髓组织中的未分化的间充质干细胞，具有高度增殖能力和多向分化潜能，它们能产生丰富的外泌体，并保留与牙髓干细胞相似的生物学特征和功能。牙髓干细胞来源外泌体与肿瘤发生发展、炎症免疫反应、牙髓组织再生、神经退行性病变等密切相关，因此牙髓干细胞外泌体在检测和治疗疾病方面具有潜在的应用价值。
巨噬细胞极化：巨噬细胞应对不同的刺激因子可以形成不同的表型，这一过程也称为巨噬细胞的再编码或表型转化。极化的巨噬细胞可以进一步影响局部免疫应答，协同各种因子调节肿瘤免疫和病原体微生物感染结局，参与免疫调节和组织损伤修复与重建过程。

背景：研究发现，在缺氧预处理条件下人牙髓干细胞能够治疗根尖周炎骨缺损，但是缺氧预处理的牙髓干细胞外泌体是否能携带母细胞的生物信息而对巨噬细胞极化产生影响尚不清楚。
目的：探讨缺氧处理牙髓干细胞来源外泌体对巨噬细胞极化的影响。
方法：将第3代牙髓干细胞放入正常培养箱和缺氧培养箱培养，培养48 h换液1次，当细胞生长至汇合率达80%-90%时收集细胞培养上清液，差速离心法提取外泌体。将人单核细胞系THP-1诱导分化为巨噬细胞，诱导的同时与PBS、正常培养和缺氧培养的牙髓干细胞来源外泌体共孵育48 h，ELISA检测细胞培养上清液中白细胞介素10 和肿瘤坏死因子α水平，RT-qPCR检测巨噬细胞CD163、白细胞介素1受体拮抗剂、转化生长因子β1、趋化因子CCL2和肿瘤坏死因子α mRNA表达，流式细胞术检测CD11b+CD163+巨噬细胞表达，Western blot检测NF-κB和STAT3信号通路激活情况。
结果与结论：①与PBS组相比，正常培养牙髓干细胞来源外泌体组巨噬细胞培养上清中白细胞介素10水平显著增加(P < 0.05)、肿瘤坏死因子α水平显著降低(P < 0.05)，巨噬细胞CD163、转化生长因子β1和趋化因子CCL2 mRNA表达均显著上升(P < 0.05)，肿瘤坏死因子α mRNA表达显著下降(P < 0.05)，CD11b+CD163+巨噬细胞阳性率显著升高(P < 0.05)，p65磷酸化水平显著降低(P < 0.05)，IκBα表达显著升高(P < 0.05)，STAT3磷酸化水平亦显著升高(P < 0.001)；②与正常培养牙髓干细胞来源外泌体组相比，缺氧培养牙髓干细胞来源外泌体组巨噬细胞上清中白细胞介素10水平显著升高(P < 0.001)，肿瘤坏死因子α水平显著降低(P < 0.01)，CD163、转化生长因子β1、趋化因子CCL2和白细胞介素1受体拮抗剂mRNA表达均显著升高(P < 0.01)，肿瘤坏死因子α mRNA表达亦显著下降(P < 0.01)，CD11b+CD163+巨噬细胞阳性率显著升高(P < 0.01)，p65磷酸化水平显著降低(P < 0.01)，IκBα表达显著升高(P < 0.01)，STAT3磷酸化水平显著升高(P < 0.01)；③结果表明，正常培养和缺氧培养牙髓干细胞来源外泌体均可促进巨噬细胞M2型极化，且缺氧培养条件下巨噬细胞M2极化更为明显，其机制可能为激活STAT3信号通路，抑制NF-κB信号通路。

https://orcid.org/0000-0001-8405-2382 (谭旭) ；https://orcid.org/0000-0003-3519-4485 (廖健) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 干细胞, 牙髓干细胞, 缺氧, 外泌体, 巨噬细胞, 极化, STAT3, NF-κB, 信号通路

Abstract: BACKGROUND: Studies have found that human dental pulp stem cells can treat periapical periodontitis bone defects under hypoxia preconditioning, but whether dental pulp stem cell exosomes under hypoxia preconditioning can carry the biological information of blasts and affect polarization of macrophages is unclear.  
OBJECTIVE: To study the effect of hypoxia-treated dental pulp stem cells exosomes on macrophage polarization.
METHODS:  Passage 3 dental pulp stem cells were cultured in a normal and hypoxic incubator. The medium was replaced once every 48 hours. Cell culture supernatants were collected at the confluence of 80%-90%. Exosomes were extracted by differential centrifugation. Human monocyte cell line THP-1 was induced to differentiate into macrophages, and co-incubated with PBS, normal culture, and hypoxic cultured dental pulp stem cells for 48 hours. Levels of inflammatory factors interleukin 10 and tumor necrosis factor alpha in supernatant were detected using ELISA. RT-qPCR was utilized to detect macrophage CD163, interleukin 1 receptor antagonist, transforming growth factor β1, chemokine CCL2, and tumor necrosis factor α mRNA expression. Flow cytometry was utilized to detect the expression of CD11b+CD163+ macrophages. Western blot assay was used to detect the activation of NF-κB and STAT3 signaling pathways.  
RESULTS AND CONCLUSION: (1) Compared with the PBS group, the level of interleukin-10 in the supernatant of macrophages in the normal cultured dental pulp stem cell-derived exosomes group significantly increased (P < 0.05), and the level of tumor necrosis factor alpha significantly reduced (P < 0.05). The expression of CD163, transforming growth factor β1, and chemokine CCL2 mRNA in macrophages increased significantly (P < 0.05); the expression of tumor necrosis factor alpha mRNA decreased significantly (P < 0.05), and the positive rate of CD11b+CD163+ macrophages increased significantly (P < 0.05); the phosphorylation level of p65 significantly reduced (P < 0.05); the expression of IκBα significantly increased (P < 0.05), and the phosphorylation level of STAT3 also significantly increased (P < 0.001). (2) Compared with the normal cultured dental pulp stem cell-derived exosomes group, the level of interleukin-10 in the supernatant of macrophages significantly increased in the hypoxic cultured dental pulp stem cell-derived exosomes group (P < 0.001), and the level of tumor necrosis factor alpha significantly reduced (P < 0.01); CD163, transforming growth factor β1, chemokine CCL2 and interleukin 1 receptor antagonist mRNA expression significantly increased (P < 0.01); tumor necrosis factor alpha mRNA expression also significantly decreased (P < 0.01); the positive rate of CD11b+CD163+ macrophages significantly increased (P < 0.01); the phosphorylation level of p65 significantly reduced (P < 0.01); the expression of IκBα significantly increased (P < 0.01), and the phosphorylation level of STAT3 significantly increased (P < 0.01). (3) It is concluded that normal culture and hypoxic culture of exosomes derived from dental pulp stem cells can promote the M2 polarization of macrophages, and the M2 polarization of macrophages is more obvious under hypoxic culture, and its mechanism may be to activate the STAT3 signaling pathway and inhibit the NF-κB signaling pathway.

Key words: stem cells, dental pulp stem cells, hypoxia, exosomes, macrophages, polarization, STAT3, NF-κB, signaling pathway

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