中国实用口腔科杂志

中国实用口腔科杂志

• 口腔颌面部组织再生专栏 • 上一篇    下一篇

骨髓间充质干细胞来源的外泌体促进牙周再生的体外研究

朱斌1，2 ，李楠1 ，田自锋1 ，张红梅3 ，刘世宇2   

  1. 1.西藏军区总医院口腔科，西藏 拉萨 850007；2. 军事口腔医学国家重点实验室，第四军医大学组织工程研发中心，陕西  西安  710032；3.第四军医大学唐都医院烧伤整形科，陕西 西安  710038
  • 出版日期:2016-12-15 发布日期:2017-01-17
  • 基金资助:

    国家自然科学基金（81601620）；中国博士后基金（2015M572684）；国家自然科学基金（81500857）

  • Online:2016-12-15 Published:2017-01-17

摘要：

目的    研究人骨髓间充质干细胞（human bone marrow mesenchymal stem cells， hBMMSCs）分泌的外泌体（exosome）对人牙周膜干细胞（human periodontal ligament stem cells， hPDLSCs）增殖和分化功能的影响，为进一步明确hBMMSCs促进牙周组织再生的机制提供实验基础和证据。方法    分别用酶消化法和离心法培养hPDLSCs和hBMMSCs，并用流式细胞术检测其间充质表面标志物。收集培养的第3代hBMMSCs上清液，利用试剂盒提取exosome，电镜下观察其结构，Western Bolt法检测其CD63表达水平。用hBMMSCs分泌的exosome处理hPDLSCs设为实验组，四甲基偶氮唑盐（MTT）法检测hPDLSCs的增殖情况，克隆形成率检测多克隆形成能力，成骨诱导后碱性磷酸酶（ALP）染色和茜素红染色并定量，同时实时荧光定量PCR（qPCR）检测成骨相关基因表达。结果    流式细胞术检测，hPDLSCs阳性表达CD29、CD44、CD90、CD146和Stro-1，hBMMSCs阳性表达CD29、CD44、CD90和 CD106，两者均阴性表达CD14、CD34和CD45。hBMMSCs分泌的exosome电镜下呈小球状，Western Bolt法检测其强表达CD63。实验组hPDLSCs经MTT法检测其增殖速率和克隆形成能力显著高于对照组（P < 0.05）。实验组hPDLSCs成骨诱导后ALP染色、茜素红染色定量表达显著强于对照组（P < 0.05）。qPCR检测成骨基因Runx2、OCN、ALP、BSP和Col-Ⅰ的表达，实验组显著高于对照组（P < 0.05）。结论    hBMMSCs分泌的exosome可促进牙周膜干细胞的体外增殖和成骨分化能力。

关键词: 人骨髓间充质干细胞, 人牙周膜干细胞, 外泌体, 成骨分化, 牙周再生

Abstract:

Objective    To study the influence of exosome secreted by the human bone marrow mesenchymal stem cells （hBMMSCs） on proliferation and differentiation of periodontal ligament stem cells （hPDLSCs），and to provide experimental basis and evidence of the mechanism of hBMMSCs in promoting periodontal tissue regeneration. Methods    hPDLSCs were cultured by enzyme digestion method and hBMMSCs were cultured by centrifugation method， and the immunophenotype was identified by flow cytometry. Supernatant of hBMMSCs in P3 was collected， exosome was extracted by kit and observed by SEM，and CD63 level was tested by WB. The experiment group was treated by exosome of hBMMSCs and control group was treated by DD water. hPDLSCs proliferation was tested by MTT and colony unite forming was tested in 7 days. After osteogenetic induction， ALP and Alizarin Red staining were performed and quantitative analysis was carried. Meanwhile osteogenesis-related gene expression was tested by qPCR. Results    hPDLSCs positively expressed CD29， CD44， CD90， CD146 and Stro-1； hBMMSCs positively expressed CD29， CD44， CD90 and CD106；both negatively expressed CD14， CD34 and CD45. Exosome secreted by hBMMSCs were small ball-shaped under SEM and expressed CD63 strongly by WB. The proliferation and colony unite forming of experimental group was significantly higher than control group （P < 0.05）. The quantitative expression of ALP and alizarin red staining after osteogenetic induction were significantly stronger than the control group （P < 0.05）. The osteogenetic gene expression，including Runx2， OCN， ALP， BSP and Col - Ⅰ， was significantly higher than that of control group by qPCR （P < 0.05）. Conclusion    The exosome secreted by hBMMSCs can promote　proliferation and osteogenesis of hPDLSCs in vitro.

Key words: human bone marrow mesenchymal stem cells, human periodontal ligament stem cells, exosome, osteogenetic differentiation, periodontal regeneration