施旺细胞衍生的细胞外囊泡通过Wnt/β-catenin信号通路促进牙髓再生的机制研究  

Extracellular vesicles derived from Schwann cells promote pulp regeneration through the Wnt/β-catenin signaling pathway

作　　者：于倩 崔庆超 范一卉 姚瑶 Yu Qian;Cui Qingchao;Fan Yihui;Yao Yao(Department of Stomatology,Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine,Cangzhou 061000,China)

机构地区：[1]河北省沧州中西医结合医院口腔科门诊,沧州061000

出　　处：《中华细胞与干细胞杂志(电子版)》2025年第1期1-11,共11页Chinese Journal of Cell and Stem Cell(Electronic Edition)

基　　金：河北省卫生健康委员会医学科学研究课题(20232144)。

摘　　要：目的探究施旺细胞衍生的细胞外囊泡(SCs-EVs)对牙髓再生的影响及其可能机制。方法体外分离培养人牙髓干细胞(hDPSCs),分别用SCs-EVs、SCs-EVs联合Wnt/β-catenin信号通路抑制剂IWR-1处理(SCs-EVs组、SCs-EVs+IWR-1组),另设置空白对照组。CCK-8法检测细胞增殖活性,平板克隆法检测细胞克隆形成能力,茜素红染色法检测细胞钙结节形成能力,油红O染色法检测细胞中脂质形成能力,RT-qPCR法检测细胞中自我更新标志基因(Oct4、Nanog、Sox2)、成骨分化标志基因(ALP、Runx2、COL-1)、成脂分化标志基因(ADPN、FABP4、LPL)和成神经分化标志基因(Gfap、Nestin、Tubb3)mRNA表达水平,Western blot法检测细胞中Runx2、ADPN、Nestin及Wnt/β-catenin信号通路蛋白表达水平,免疫荧光检测β-catenin荧光强度。两组间比较采用独立样本t检验或校正t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验(不同SCs-EVs浓度组间;SCs-EVs组与Control组;SCs-EVs+IWR-1组与SCs-EVs组)。结果与空白对照相比,SCs-EVs组细胞增殖活性增强(P<0.05),同时,克隆团数量(909.17±52.86比415.38±25.61)、钙结节形成能力(0.79±0.05比0.24±0.02)、脂质形成能力(0.52±0.03比0.14±0.01),Oct4、Nanog、Sox2、ALP、Runx2、COL-1、ADPN、FABP4、LPL、Gfap、Nestin和Tubb3 mRNA表达水平,Runx2(1.05±0.16比0.18±0.05)、ADPN(0.91±0.09比0.14±0.04)、Nestin(0.96±0.13比0.15±0.03)和β-catenin(1.01±0.12比0.19±0.03)蛋白表达水平及β-catenin荧光强度(4.27±0.31比1.07±0.12)均升高(P均<0.05)。与SCs-EVs组比较,SCs-EVs+IWR-1组细胞增殖活性减弱,同时,克隆团数量(482.55±29.32比909.17±52.86)、钙结节形成能力(0.32±0.03比0.79±0.05)、脂质形成能力(0.20±0.01比0.52±0.03),Oct4、Nanog、Sox2、ALP、Runx2、COL-1、ADPN、FABP4、LPL、Gfap、Nestin和Tubb3 mRNA表达水平,Runx2(0.21±0.05比1.05±0.16)、ADPN(0.37±0.06比0.91±0.09)、Nestin(0.34±0.09比0.96±0.13)和β-catenin(0.31±0.Objective To investigate the effects and mechanisms of Schwann cells-derived extracellular vesicles(SCs-EVs)on pulp regeneration.Methods Human dental pulp stem cells(hDPSCs)were isolated and cultured in vitro,and treated with SCs-EVs,SCs-EVs combined with Wnt/β-catenin signaling pathway inhibitor IWR-1(SCs-EVs group,SCs-EVs+IWR-1 group),and a blank treatment group(Control group)was set up.The cell proliferative activity and cell clonogenesis ability were detected by CCK-8 method and plate cloning method respectively.The formation ability of calcium nodules was detected by alizarin red staining method.The lipid formation capacity in cells was detected by oil red O staining method.The mRNA expression levels of self-renewal marker genes(Oct4,Nanog,Sox2),osteogenic differentiation marker genes(ALP,Runx2,COL-1),lipogenic differentiation marker genes(ADPN,FABP4,LPL)and neurogenic differentiation marker genes(Gfap,Nestin,Tubb3)were detected by RT-qPCR.The protein expression level of Runx2,ADPN,Nestin and Wnt/β-catenin signaling pathway related proteinswere detected by Western blot.The fluorescence intensity ofβ-catenin was detected by immunofluorescence.Independent sample t test or corrected t test was used for comparison between two groups,one-way ANOVA was used for comparison between multiple groups,and LSD-t test was used for further pair-to-group comparison(between groups with different SCs-EVs concentrations;SCs-EVs group and Control group;SCs-EVs+IWR-1 group and SCs-EVs grou).Results Compared with the Control group,the cell proliferation activity in SCs-EVs group was enhanced(P<0.05),together with an increasing of the number of clones(909.17±52.86 vs 415.38±25.61),calcium nodule formation ability(0.79±0.05 vs 0.24±0.02),lipid formation ability(0.52±0.03 vs 0.14±0.01),Oct4,Nanog,Sox2,ALP,Runx2,COL-1,ADPN,FABP4,LPL,Gfap,Nestin,Tubb3 mRNA expression levels,Runx2(1.05±0.16 vs 0.18±0.05),ADPN(0.91±0.09 vs 0.14±0.04),Nestin(0.96±0.13 vs 0.15±0.03),β-catenin(1.01±0.12 vs 0.19±0.03)protein expression lev

关 键 词：施旺细胞 细胞外囊泡 牙髓再生 牙髓干细胞 增殖 多能性 

分 类 号：R73[医药卫生—肿瘤]