线粒体转移对牙源性间充质干细胞成牙向分化影响的研究  

Mitochondrial transfer contributes to the odontogenic differentiation of dental mesenchymal stem cells

作　　者：李欣雨 张蕾 孙瑶[1] Li Xinyu;Zhang Lei;Sun Yao(Department of Implantology,Stomatological Hospital and Dental School,Tongji University&Shanghai Engineering Research Center of Tooth Restoration and Regeneration&Tongji Research Institute of Stomatology,Shanghai200072,China)

机构地区：[1]同济大学附属口腔医院种植科,同济大学口腔医学院,上海牙组织修复与再生工程技术研究中心,同济大学口腔医学研究所,上海200072

出　　处：《中华口腔医学杂志》2025年第1期43-53,共11页Chinese Journal of Stomatology

基　　金：国家自然科学基金(82270963,U23A20444)。

摘　　要：目的探究牙源性间充质干细胞间是否存在线粒体转移以及线粒体转移对其成牙向分化的影响。方法流式细胞术分选胚胎13~14 d小鼠下颌第一磨牙牙胚间充质细胞, 得到牙源性间充质干细胞, 通过免疫组织化学染色明确牙源性间充质干细胞属性。通过免疫荧光染色及活细胞成像检测牙源性间充质干细胞间是否存在线粒体转移;分析基因表达数据库中的人牙源性间充质干细胞与骨髓间充质干细胞转录组测序数据, 探究线粒体相关基因在两组样本间是否存在差异。以二甲基亚砜作为对照组, 使用不同浓度(1、5、10 μmol/L)线粒体转移抑制剂ML141对原代细胞进行处理, 利用鬼笔环肽免疫荧光染色、细胞活性氧检测、衰老相关β-半乳糖苷酶(SA-β-gal)染色、5-乙炔-2′-脱氧尿苷(EdU)、细胞计数试剂盒(CCK-8)实验、蛋白质印迹法、活细胞成像以及透射电镜观察间充质干细胞间线粒体转移被抑制后对细胞形态、细胞衰老、细胞活性氧、细胞增殖、间充质干细胞标志物配对相关同源基因1(Prrx1)和成牙分化相关基因Sp7转录因子(Sp7)蛋白水平、线粒体转移水平以及线粒体形态的影响。进一步在成牙向分化诱导过程中使用线粒体转移抑制剂, 通过碱性磷酸酶(ALP)显色试剂盒检测ALP活性, 实时荧光定量PCR(RT-qPCR)检测成牙分化相关基因Alp、Sp7、牙本质基质蛋白1(Dmp1)和牙本质涎磷蛋白(Dspp)表达, 观察抑制牙源性间充质干细胞间线粒体转移对其成牙向分化能力的影响。结果牙源性间充质干细胞间存在细长形态、被F-肌动蛋白(F-actin)标记的隧道纳米管(TNTs)结构, 同时观察到该结构中存在正在转移的线粒体。转录组测序数据显示, 牙源性间充质干细胞和骨髓间充质干细胞基因表达谱特征存在明显区别, 牙源性间充质干细胞特异性上调线粒体转移及线粒体动力学相关基因。相较于对照组Objective To investigate whether there is mitochondrial transfer in dental mesenchymal stem cells(MSCs)and its significance for the odontogenic differentiation.MethodsFlow cytometry and immunohistochemical staining were used to isolate dental mesenchymal stem cells.Immunofluorescence staining and live cell imaging were applied to determine whether there is mitochondrial transfer in dental MSCs.Transcriptome sequencing data re-analysis of human dental pulp stem cells(DPSCs)and bone marrow mesenchymal stem cells(BMSCs)from gene expression omnibus(GEO)data base demonstrated the importance of mitochondrial transfer in dental MSCs.Cells were managed with mitochondrial transfer inhibitor ML141 with dimethyl sulfoxide as the control.Immunofluorescence staining,senescence-associatedβ-galactosidase(SA-β-gal)staining,reactive oxygen species(ROS)assay,5-ethynyl-2'-deoxyuridine(EdU)labelling,cell counting kit-8(CCK-8)assay,Western blotting,live cell imaging and transmission electron microscope were used to investigate cell morphology,ROS level,cellular senescence,cell proliferation,MSCs marker paired related homeobox 1(Prrx1)and Sp7 transcription factor(Sp7)expression,mitochondrial transfer and mitochondrial morphology,respectively.Further,after using ML141 during the induction of odontogenic differentiation,alkaline phosphatase(ALP)chromogenic kit was used to detect ALP activity and real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of odontogenic differentiation-related genes Alp,Sp7,dentin matrix protein 1(Dmp1),and dentin salivary phosphoprotein(Dspp),which were applied to investigate the effect of mitochondrial transfer on odontogenic differentiation.ResultsAn ultrafine tunneling nanotubes(TNTs)structure labelled with F-actin existed between dental MSCs,and the presence of transferring mitochondria in this structure was also confirmed.Transcriptome sequencing data suggested that the gene expression profiles were significantly different between DPSCs and BMSCs.Genes related to mitochondri

关 键 词：线粒体 间充质干细胞 牙源性间充质干细胞 线粒体转移 细胞分化 成牙分化 

分 类 号：R73[医药卫生—肿瘤]