口腔医学研究

口腔医学研究 ›› 2019, Vol. 35 ›› Issue (1): 38-41.DOI: 10.13701/j.cnki.kqyxyj.2019.01.009

• 牙周病学研究 • 上一篇    下一篇

低氧对人牙周膜干细胞增殖和分化的影响

郑树灿, 邹晖, 黄旭瑶, 邵海宾, 周勇, 张瑞芳, 李朝晖*   

  1. 广州市花都区妇幼保健院(胡忠医院),广东医科大学附属广州花都医院口腔治疗中心 广东 广州 510800
  • 收稿日期:2018-07-07 出版日期:2019-01-18 发布日期:2019-01-28
  • 通讯作者: 李朝晖,E-mail: gdlzh1013@163.com
  • 作者简介:郑树灿(1983~ ),男,广东潮州人,硕士,主治医师,主要从事口腔修复学和口腔种植学研究。
  • 基金资助:
    广东省自然科学基金(编号:2013B021800076)

Effect of Hypoxia on Proliferation and Differentiation of Human Periodontal Ligament Stem Cells

ZHENG Shu-can, ZOU Hui, HUANG Xu-yao, SHAO Hai-bin, ZHOU Yong, ZHANG Rui-fang, LI Chao-hui*   

  1. Stomatology Center, Guangzhou Huadu District Maternal and Child Health Hospital; Affiliated Guangzhou Huadu Hospital, Guangdong Medical University, Guangzhou 510800, China
  • Received:2018-07-07 Online:2019-01-18 Published:2019-01-28

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摘要： 目的:观察低氧预处理对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)干性的影响。方法:收集hPDLSCs并进行鉴定后,分别在常氧(20%O2)和低氧(5%O2) 环境下培养5 d,噻唑蓝(methyl thiazol tetrazolium,MTT)法检测细胞的增殖能力。分别在低氧环境培养1 d和5 d,实时定量聚合酶链反应(Real-time quantitative polymerase chain reaction,Real-time PCR)检测hPDLSCs中干性相关基因Oct4、Sox2、Nanog的mRNA表达水平。细胞增殖核抗原Ki-67染色法和结晶紫染色观察细胞自我更新能力。分别用成骨和成脂诱导hPDLSCs 21 d,观察低氧对人牙周膜干细胞多项分化能力的影响。结果:MTT结果显示低氧预处理能显著增强hPDLSCs增殖能力(P<0.05)。Real-time PCR结果显示低氧在第1天和第5天时能够提高细胞的干性基因表达(P<0.05),其中在第5天的时候SOX2的提高最为显著(P<0.05)。结晶紫染色结果表明,低氧环境能够增加细胞克隆形成率(P<0.05)。低氧环境还能增强干细胞的成骨分化能力,成骨相关基因RUNX2的表达显著提高(P<0.05),但是对成脂相关基因的增强不明显,对成脂标志基因PPARγ2的影响不明显。结论:低氧预处理hPDLSCs能够提高细胞的增殖和干性相关基因的表达。另外,低氧环境也能提高干细胞的分化,其中对成骨相关基因的促进作用较为显著,但对成脂分化影响不大。

关键词: 低氧, 人牙周膜干细胞, 增殖, 分化

Abstract: Objective: To examine the effect of hypoxia on human periodontal ligament stem cells (hPDLSCs). Methods: HPDLSCs were collected and validated. Cells were cultured in normoxia (20% O2) or hypoxia (5%O2) for 5 days. MTT was applied to detect the cell proliferation ability in each day. OCT4, SOX2, and NANOG genes were analyzed by RT-PCR in cells culture in hypoxia at day 1 and day 5. Ki67 staining and crystal violet staining assay were used to detect the self-renewal ability of hPDLSCs. Osteogenic and adipogenic differentiation abilities were investigated after 21-day-induction. Results: The results from MTT assay showed that the proliferation ability of hPDLSCs treated with hypoxia was higher (P<0.05). Hypoxia treatment enhanced the related gene expression at both day 1 and day 5 (P<0.05). SOX2 was enhanced strongly at day 5 (P<0.01). Crystal violet staining assay indicated that colony-forming efficiency was increased in hypoxia group (P<0.05). In addition, hypoxia also enhanced the osteogenic differentiation capacity of hPDLSCs. Osteogenic gene RUNX2 was significantly increased (P<0.05). However, although hypoxia increased the expression of adipogenic gene PPAR γ2 and adipogenic differentiation, the results was not statistically significant. Conclusion: Hypoxic preconditioning of hPDLSCs can increase cell proliferation and expression of stemness-related genes. In addition, hypoxia environment can also improve the osteogenesis differentiation of hPDLSCs, but has little effect on adipogenic differentiation.

Key words: Hypoxia, Human periodontal ligament stem cells, Proliferation, Differentiation