为了探究来自牙髓干细胞（DPSCs）的外泌体在牙源性分化方向上的特征，分析来自未分化和牙源性DPSCs的外泌体中microRNA表达谱的差异，并分析它们可能的信号转导途径。（1）用α最小的老鹰培养基（α-MEM）培养DPSCs，并用牙源性分化培养基培养牙源性DPSCs 21天，用茜素红染色和碱性磷酸酶染色鉴定牙源性分化。分别从细胞上清液中分离出外泌体，命名为牙髓干细胞外泌体（DPSCs-Exo）和牙髓干细胞牙源性外泌体（DPSCs-OD-Exo）。通过透射电镜、纳米粒子跟踪分析和西方印迹验证外泌体。（2）通过microRNA微阵列研究DPSCs-Exo和DPSCs-OD-Exo的microRNA表达谱。为验证microRNA微阵列的结果，采用实时定量聚合酶链反应（实时PCR）对3个最显著差异表达的microRNA进行验证。通过通路分析检测与microRNA预测靶基因相关的富集通路。（1）离体培养的DPSCs呈纤维母细胞状典型形态。牙源性分化的DPSCs呈纺锤形、多角形、大小均匀。牙源性分化组在茜素红染色中有大量黑色沉积物，碱性磷酸酶染色中细胞染色较深，而正常培养基组细胞没有明显染色。DPSCs-Exo和DPSCs-OD-Exo具有相同的形态，均呈双层膜和杯状。DPSCs-Exo和DPSCs-OD-Exo的峰值大小分别为（114.67±9.07）nm和（134.00±8.54）nm，两者之间差异显著。DPSCs-Exo和DPSCs-OD-Exo均表达外泌体标记物肿瘤易感基因（TSG）101和CD63。（2）microRNA微阵列结果显示DPSCs-Exo和DPSCs-OD-Exo的表达谱不同。其中19个增加了两倍以上，一个减少了64%。实时PCR结果显示，在DPSCs-OD-Exo中，microRNA-1246、microRNA-1246-100-5p和microRNA-1246-494-3p的表达水平显著上调。差异有统计学意义。使用microRNA靶基因预测数据库和基因信号通路数据库分析差异表达的microRNA，预测差异表达的microRNA可能靶向轴抑制蛋白2（AXIN2）基因和Wnt/β-连环蛋白信号通路。DPSCs-OD-Exo和DPSCs-Exo在其microRNA表达谱中存在差异。这些差异表达的microRNA可能参与调节DPSCs的牙源性分化。

To investigate the characteristics of exosomes derived from dental pulp stem cells (DPSCs) in the direction of odontogenic differentiation, to analyze the differences in microRNA expression profile between exosomes derived from undifferentiated and odontogenic DPSCs, and to analyze their possible signal transduction pathways.(1) DPSCs were cultured in α minimum Eagle' s medium (α-MEM), and odontogenic DPSCs were cultured in odontogenic differentiation medium for 21 days, using alizarin red staining and alkaline phosphatase staining to identify the odontogenic differentiation. Exosomes from the cell supernatant were isolated respectively, named as dental pulp stem cells-exosomes (DPSCs-Exo) and dental pulp stem cells-odontogenic-exosomes (DPSCs-OD-Exo). The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and Western blot. (2) The microRNA expression profiles of DPSCs-Exo and DPSCs-OD-Exo were investigated by microRNA microarray. To validate the result of the microRNA microarray, real-time quantitative polymerase chain reaction (real-time PCR) assay was applied on 3 most significantly differential expressed microRNA. Pathway analysis was taken to detect enriched pathways associated with the predicted target genes of microRNA.(1) The DPSCs were isolated and cultured in vitro showed typical fibroblast-like morphology. The odontogenic differentiated DPSCs were spindle-shaped, polygonal, and uniform in size. Odontogenic differentiation group showed a large number of dark deposits in alizarin red staining and the cells were darkly stained in alkaline phosphatase staining, while the cells in normal culture medium group did not show obvious dyeing. The DPSCs-Exo and DPSCs-OD-Exo had the same morphology, both showed bilayer membrane and cup-shape. The peak sizes of DPSCs-Exo and DPSCs-OD-Exo were (114.67±9.07) nm and (134.00±8.54) nm, respectively. The difference between the two was statistically significant. DPSCs-Exo and DPSCs-OD-Exo both expressed the markers of exosomes, tumor susceptibility gene (TSG)101 and CD63. (2) microRNA microarray results showed that the expression profiles of DPSCs-Exo and DPSCs-OD-Exo were different. Nineteen increased by more than two times, and one decreased by 64%. Real-time PCR results showed that the expression levels of microRNA-1246, microRNA-1246-100-5p and microRNA-1246-494-3p in DPSCs-OD-Exo were significantly up-regulated. The difference was statistically significant. microRNA target prediction database and gene signaling pathway database were used to analyze differentially expressed microRNA, and it was predicted that differentially expressed microRNA could target axis inhibition protein 2(AXIN2) gene and Wnt/β-catenin signaling pathway.DPSCs-OD-Exo and DPSCs-Exo had differences in their microRNA expression profile. Those differentially expressed microRNA may be involved in the regulation of DPSCs odontogenic differentiation.