中国实用口腔科杂志

中国实用口腔科杂志

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牵张力对牙周膜干细胞内质网线粒体偶联及其介导成骨分化能力的影响研究

杨宽1，2，包立辉2，张彩娣3，翟启明2，邸天凯3，王爽1   

  1. 1. 西安交通大学口腔医学院正畸科，陕西  西安 710004；2. 第四军医大学口腔医学院组织工程中心，陕西  西安 710032；3. 军事口腔医学国家重点实验室，口腔疾病国家临床医学研究中心，陕西省口腔疾病临床医学研究中心，第四军医大学口腔医院儿童口腔科，陕西  西安  710032
  • 出版日期:2021-05-30 发布日期:2021-06-03
  • 基金资助:
    国家自然科学基金（81670988）；国家口腔疾病临床医学研究中心资助课题（LCA202010）；军事口腔医学国家重点实验室自研课题（2019ZA06）

  • Online:2021-05-30 Published:2021-06-03

摘要： 目的    探究牙周膜干细胞（periodontal ligament stem cells，PDLSCs）在受到牵张力作用下的成骨分化能力及其内质网线粒体偶联的变化。方法    从牙周膜分离原代PDLSCs并进行传代培养，使用第3代细胞以施加或不施加牵张力处理分别作为实验组和对照组，并在加力后收取细胞进行成骨诱导培养，采用茜素红染色和实时荧光定量PCR（qRT-PCR）检测PDLSCs成骨分化水平和成骨相关基因［Runt相关转录因子2（Runx2）、骨钙素（OCN）、碱性磷酸酶（ALP）］的表达水平。用透射电镜观察线粒体内质网的空间接触变化，采用qRT-PCR检测线粒体融合蛋白-1（mitofusion 1，Mfn1）和线粒体融合蛋白-2（mitofusion 2，Mfn2）的基因表达水平，采用线粒体膜电位检测试剂盒检测PDLSCs线粒体膜电位变化。结果    茜素红染色和qRT-PCR检测结果显示，施加牵张力后PDLSCs的成骨分化能力显著增强（P < 0.05）。透射电镜结果显示，与对照组相比，实验组PDLSCs内质网线粒体偶联长度与线粒体周长之比显著降低（P < 0.05）。实验组细胞Mfn2基因表达水平较对照组显著降低（P < 0.05），但两组Mfn1基因表达变化差异无统计学意义（P > 0.05）。实验组细胞线粒体膜电位与对照组相比则显著升高（P < 0.05）。结论    施加牵张力可促进PDLSCs的成骨分化，其机制可能与抑制PDLSCs内质网线粒体的偶联、升高线粒体膜电位趋势有关。

关键词: 牙周膜干细胞, 牵张力, 内质网线粒体偶联, 线粒体膜电位, 成骨分化

Abstract: Objective    To investigate the osteogenic differentiation ability and the changes in endoplasmic reticulum-mitochondrial contact of periodontal stem cells（PDLSCs）under the action of tension. Methods    PDLSCs were isolated from periodontal membrane and cultured. The third generation of cells were used as experimental group and control group according to whether there was tension or not respectively. After the force was applied，the cells were collected for osteoblast induction culture. The osteogenic differentiation level and the expression of osteogenic genes of PDLSCs ［Runt-related transcription factor 2（Runx2），osteocalcin （OCN） and alkaline phosphatase （ALP）］ were detected by alizarin red staining and real-time fluorescence quantitative PCR（qRT-PCR）. The changes in endoplasmic reticulum-mitochondrial space contact were observed by transmission electron microscope. The expression levels of mitofuson 1（Mfn1）and mitofusion-2（Mfn2）were detected by qRT-PCR. The mitochondrial membrane potential of PDLSCs was detected by using mitochondrial membrane potential detection kit. Results    The results of alizarin red staining and qRT-PCR showed that PDLSCs had a significant increase in osteogenic differentiation after tension（P < 0.05）. The results of transmission electron microscopy showed that the ratio of the coupling length of PDLSCs mitochondria to the perimeter of mitochondria in experimental group was significantly lower than that in the control group（P < 0.05）. The expression level of Mfn2 gene in the experimental group was significantly lower than that in the control group（P < 0.05），but the difference in Mfn1 expression between the two groups was not statistically significant（P > 0.05）. The mitochondrial membrane potential of the experimental group was significantly higher than that of the control group（P < 0.05）. Conclusion    Tension can promote the osteogenic differentiation of PDLSCs，and its mechanism may be related to the inhibition of endoplasmic reticulum-mitochondrial contact and the increase of mitochondrial membrane potential.

Key words: periodontal ligament stem cells, tension, endoplasmic reticulum-mitochondrial contact, mitochondrial membrane potential, osteogenic differentiation