口腔疾病防治 ›› 2020, Vol. 28 ›› Issue (9): 569-574.DOI: 10.12016/j.issn.2096-1456.2020.09.004

• 基础研究 • 上一篇    下一篇

miR-21对人牙周膜干细胞增殖及成骨分化的影响

马灵芝(),施娇壮,戈文斌,张琨,余兵,刘亚丽()   

  1. 昆明医科大学附属口腔医院口腔正畸科,云南 昆明(650101)
  • 收稿日期:2019-12-25 修回日期:2020-02-03 出版日期:2020-09-20 发布日期:2020-08-24
  • 通讯作者: 刘亚丽
  • 作者简介:马灵芝,医师,硕士研究生在读,Email:422385890@qq.com
  • 基金资助:
    国家自然科学基金资助项目(31860326);云南省应用基础研究计划项目(2017FE468-227);云南省应用基础研究计划项目(2018FE001-260);昆明医科大学研究生创新基金资助项目(2019S018)

Effect of miR-21 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells

MA Lingzhi(),SHI Jiaozhuang,GE Wenbin,ZHANG Kun,YU Bing,LIU Yali()   

  1. Department of Orthodontics, the Affiliated Stomatological Hospital of Kunming Medical University, Yunnan 650101, China
  • Received:2019-12-25 Revised:2020-02-03 Online:2020-09-20 Published:2020-08-24
  • Contact: Yali LIU

摘要:

目的 探讨miR-21对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)增殖及成骨的影响,旨在为干细胞治疗牙周炎提供实验基础。方法 酶解组织块法分离培养hPDLSCs,通过流式细胞仪检测表面分子CD34、CD45、CD90、CD105对hPDLSCs进行鉴定,采用Lipofectamine 2000将 miR-21的mimics(pre-miR-21)、inhibitor(anti-miR-21)以及各自的阴性对照转染至hPDLSCs。实验分组:过表达组(mimics组)、过表达阴性对照组(mimics-NC组);抑制阴性对照组(inhibitor-NC组)、抑制组(inhibitor组)。实时荧光定量PCR检测miR-21转染效率;CCK-8、流式细胞术检测hPDLSCs的增殖;茜素红染色检测hPDLSCs的成骨能力和Western blot检测成骨相关基因Runx2的蛋白表达。结果 miR-21的mRNA表达量mimics组较mimics-NC组明显升高,inhibitor组较inhibitor-NC组明显降低(P < 0.05)。hPDLSCs增殖及S期细胞比例mimics组较mimics-NC组升高,inhibitor组较inhibitor-NC组减少,差异有统计学意义(P < 0.05)。经茜素红染色后,mimics组矿化结节多于mimics-NC组;inhibitor组矿化结节少于inhibitor-NC组;Western blot检测mimics组Runx2蛋白表达高于mimics-NC组,inhibitor组Runx2蛋白表达量较inhibitor-NC组减少,差异有统计学意义(P<0.05)。结论 miR-21调控hPDLSCs的增殖和成骨向分化。

关键词: 微小RNA, miR-21, 牙周再生, 人牙周膜干细胞, 增殖, 成骨分化, 矿化结节, 成骨相关转录因子2

Abstract:

Objective To explore the effect of miR-21 on human periodontal ligament stem cells (PDLSCs) proliferation and osteogenesis and to provide a theoretical basis for the stem cell treatment of periodontitis.Methods hPDLSCs were isolated and cultured with the enzymatic tissue block method, and surface molecules (CD34, CD45, CD90 and CD105) were detected by flow cytometry. An miR-21 mimics (pre-miR-21) and inhibitor (anti-miR-21) were transfected into hPDLSCs by Lipofectamine 2000. The experiment groups: mimics-NC group, mimics group, inhibitor group, and inhibitor-NC group. The transfection efficiency of miR-21 was determined by qRT-PCR. Proliferation was detected by CCK-8 and flow cytometry. The osteogenic differentiation ability of hPDLSCs was determined by alizarin red staining. Western blot was used to detect the protein expression of osteogenic related genes: Runx2.Results The mRNA expression of miR-21in the mimics group was significantly higher than that in the mimics-NC group; additionally, the expression in the inhibitor group was significantly weaker than that in the inhibitor-NC group (P < 0.05). hPDLSCs proliferation and the S phase cell ratio in the mimics group were stronger than those in the mimics-NC group(P < 0.05); those in the inhibitor group were weaker than those in the inhibitor-NC group (P < 0.05). After alizarin red staining, the mimics group was found to have more mineralized modules than mimics-NC group, and the inhibitor group had fewer than that in the inhibitor-NC group. Runx2 protein expression in the mimics group was higher than that in the mimics-NC group (P <0.05), and expression was lower in the inhibitor group than in the inhibitor-NC group (P < 0.05).Conclusion miR-21can promote the proliferation and osteogenesic differentiation of hPDLSCs.

Key words: microRNA, miR-21, periodontal tissue regeneration, human periodontal stem cells, proliferation, osteogenesis differentiation, mineralized module, runt-related transcription factor-2

中图分类号: