成牙本质方向分化牙髓干细胞外泌体形态及微小RNA表达谱特征

Characteristics and microRNA expression profile of exosomes derived from odontogenic dental pulp stem cells

目的:研究成牙本质方向分化下牙髓干细胞(dental pulp stem cells,DPSCs)来源外泌体的特征,比较其与普通培养下DPSCs来源外泌体的微小RNA(microRNA)表达差异,并分析其相关信号传导通路。方法:(1)分别采用α-基础伊格尔(氏)培养基(αminimum Eagle’s medium,α-MEM,Hyclone公司,美国)和成牙本质方向分化培养基培养DPSCs 21 d,使用茜素红矿化结节染色和碱性磷酸酶染色对两种细胞进行鉴定。分别在两种细胞上清液中提取外泌体,命名为普通培养条件下DPSCs外泌体(dental pulp stem cells-exosomes,DPSCs-Exo)和成牙本质方向分化培养条件下DPSCs外泌体(dental pulp stem cells-odontogenic-exosomes,DPSCs-OD-Exo)。采用透射电镜观察法、纳米粒子示踪分析法和蛋白印迹法观察比较两种外泌体的形态、粒径分布和外泌体标记蛋白表达情况。(2)采用microRNA芯片法分析DPSCs-Exo和DPSCs-OD-Exo的microRNA表达谱差异,选择差异表达最显著的3种micro-RNA进行实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,real-time PCR)验证。对差异表达的microRNA采用microRNA目标预测数据库及基因信号通路数据库进行分析,预测microRNA在DPSCs成牙本质方向分化中的信号传导途径。结果:(1)普通培养条件下的DPSCs呈梭形、多角形等典型的成纤维细胞样形态,成牙本质方向诱导分化21 d后的DPSCs也基本呈梭形、多角形。成牙本质方向诱导分化条件下的细胞茜素红矿化结节染色结果镜下可见大量色暗沉积物形成,碱性磷酸酶染色结果显示细胞颜色深染呈深蓝色,而普通培养条件下的细胞则未见明显染色。两种培养条件下的DPSCs-Exo和DPSCs-OD-Exo形态一致,均呈茶托样,具有双层膜结构。DPSCs-Exo粒径峰值为(114.67±9.07)nm,DPSCs-OD-Exo粒径峰值为(134.00±8.54)nm。DPSCs-OD-Exo的粒径峰值稍大于DPSCs-Exo,差异有统计学意义(t=58.00,P<0.05)。DPSCs-Exo和DPSCs-OD-Exo均表达外泌体标志蛋白肿瘤易感基因(tumor susceptibility gene,TSG)101蛋白、CD63,符合外泌体特征。(2)microRNA芯片结果显示DPSCs-Exo与DPSCs-OD-Exo的microRNA表达谱存在差异,其中19个增加2倍以上,1个减少64%。real-time PCR验证结果显示,microRNA表达谱中差异表达的microRNA-1246、microRNA-100-5p和microRNA-494-3p在DPSCs-Exo与DPSCs-OD-Exo中存在差异,且差异有统计学意义(P<0.05)。通过microRNA目标预测数据库及基因信号通路数据库对差异表达的microRNA进行分析,预测差异表达的microRNA可靶向轴抑制蛋白2(axis inhibition protein 2,AXIN2)基因及Wnt/β-catenin信号传导通路。结论:DPSCs-OD-Exo与DPSCs-Exo均符合外泌体特征,两者的microRNA表达谱存在差异,差异表达的microRNA可能参与调控DPSCs成牙本质方向分化。

Objective: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.Methods:(1)DPSCs were cultured inαminimum 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.Results:(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.Conclusion: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.