Li X Y, Zhang L, Sun Y
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.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2025 Jan 9;60(1):43-53. doi: 10.3760/cma.j.cn112144-20240926-00360.
To investigate whether there is mitochondrial transfer in dental mesenchymal stem cells (MSCs) and its significance for the odontogenic differentiation. Flow 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-associated β-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. An 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 mitochondrial transfer and mitochondrial dynamic were significantly increased in DPSCs compared to BMSCs. Compared with the control group, treatment with 1, 5, 10 μmol/L ML141, the mitochondrial transfer inhibitor, had little significant effects on the cell morphology, cytoskeleton and ROS level. SA-β-gal activity and the proportion of SA-β-gal positive cells in the ML141-treated groups [(3.93±0.21)%, (3.23±0.42)%, (4.06±0.84)%] had no significant differences with the control group [(3.83±0.28)%] (all >0.05). In the cell proliferation assay, the proportion of EdU positive cells in the ML141-treated groups [(20.00±3.82)%, (19.48±1.96)%, (12.55±2.86)%] had no significant differences (all >0.05) with the control group [(18.57±0.87)%], whereas the CCK-8 assay showed similar results in ML141-treated group of 1, 5 μmol/L (all >0.05). Western blotting results showed that the protein expression levels of PRRX1 and SP7 in the ML141-treated group had no significant differences with the control group. Live cell imaging showed that compared with the control group [(31.42±4.01)%], the proportion of TNTs and mitochondrial transfer in the ML141-treated groups [(13.45±1.46)%, (10.36±3.47)%, (9.32±1.11)%] were significantly decreased in dental MSCs (all <0.001). Scanning electron microscope showed that the mitochondrial morphology of dental MSCs in the ML141-treated group was similar to the control group, with globular and short-rod shape. After 7 days of odontogenic differentiation, the ALP staining intensity of the ML141-treated group was significantly lower than the control group. After 21 days of induction, RT-qPCR results showed that compared with control group, the relative mRNA expressions of Alp, Sp7, Dmp1 and Dspp were significantly decreased in the ML141-treated group (all <0.05), indicating that the suppression of mitochondrial transfer in dental MSCs inhibited the odontogenic differentiation. Mitochondrial transfer exists between dental MSCs, and inhibition of mitochondrial transfer impairs the odontogenic differentiation.
研究牙间充质干细胞（MSCs）中是否存在线粒体转移及其对成牙分化的意义。采用流式细胞术和免疫组织化学染色分离牙间充质干细胞。应用免疫荧光染色和活细胞成像确定牙间充质干细胞中是否存在线粒体转移。对来自基因表达综合数据库（GEO）的人牙髓干细胞（DPSCs）和骨髓间充质干细胞（BMSCs）的转录组测序数据进行重新分析，证明了线粒体转移在牙间充质干细胞中的重要性。用线粒体转移抑制剂ML141处理细胞，以二甲基亚砜作为对照。采用免疫荧光染色、衰老相关β-半乳糖苷酶（SA-β-gal）染色、活性氧（ROS）检测、5-乙炔基-2'-脱氧尿苷（EdU）标记、细胞计数试剂盒-8（CCK-8）检测、蛋白质印迹法、活细胞成像和透射电子显微镜分别研究细胞形态、ROS水平、细胞衰老、细胞增殖、间充质干细胞标志物配对相关同源框1（Prrx1）和Sp7转录因子（Sp7）表达、线粒体转移和线粒体形态。此外，在诱导成牙分化过程中使用ML141后，用碱性磷酸酶（ALP）显色试剂盒检测ALP活性，用实时荧光定量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）的表达，以研究线粒体转移对成牙分化的影响。牙间充质干细胞之间存在标记有F-肌动蛋白的超微隧道纳米管（TNTs）结构，并且也证实了该结构中存在转移的线粒体。转录组测序数据表明，DPSCs和BMSCs之间的基因表达谱存在显著差异。与BMSCs相比，DPSCs中与线粒体转移和线粒体动力学相关的基因显著增加。与对照组相比，用1、5、10 μmol/L线粒体转移抑制剂ML141处理对细胞形态、细胞骨架和ROS水平几乎没有显著影响。ML141处理组中SA-β-gal活性和SA-β-gal阳性细胞比例[（3.93±0.21）%，（3.23±0.42）%，（4.06±0.84）%]与对照组[（3.83±0.28）%]无显著差异（均>0.05）。在细胞增殖检测中，ML141处理组中EdU阳性细胞比例[（20.00±3.82）%，（19.48±1.96）%，（12.55±2.86）%]与对照组[（18.57±0.87）%]无显著差异（均>0.05），而CCK-8检测在1、5 μmol/L ML141处理组中显示出类似结果（均>0.05）。蛋白质印迹结果表明，ML141处理组中PRRX1和SP7的蛋白表达水平与对照组无显著差异。活细胞成像显示，与对照组[（31.42±4.01）%]相比，ML141处理组中牙间充质干细胞的TNTs和线粒体转移比例[（13.45±1.46）%，（10.36±3.47）%，（9.32±1.11）%]显著降低（均<0.001）。扫描电子显微镜显示，ML141处理组牙间充质干细胞的线粒体形态与对照组相似，呈球状和短杆状。成牙分化第7天后，ML141处理组的ALP染色强度显著低于对照组。诱导21天后，RT-qPCR结果显示，与对照组相比，ML141处理组中Alp、Sp7、Dmp1和Dspp的相对mRNA表达显著降低（均<0.05），表明抑制牙间充质干细胞中的线粒体转移会抑制成牙分化。牙间充质干细胞之间存在线粒体转移，抑制线粒体转移会损害成牙分化。
