IL-34介导的NF-кB信号通路对根尖牙乳头干细胞成牙成骨定向分化的调节作用  

Regulation function of NF-кB signaling pathway mediated by IL-34 on odontogenic/osteogenic directional differentiation of stem cells from apical papilla

作　　者：朱永娜 武峻捷 刘茜 张伟健 石昕 ZHU Yongna;WU Junjie;LIU Xi;ZHANG Weijian;SHI Xin(Department of Stomatology,The Second Affiliated Hospital of Bengbu Medical University,Bengbu,Anhui 233000,China)

机构地区：[1]安徽省蚌埠医科大学第二附属医院口腔科,233000

出　　处：《淮海医药》2024年第4期331-336,共6页Journal of Huaihai Medicine

基　　金：蚌埠医学院自然科学重点项目(2021byzd192);安徽省高校自然科学研究重点项目(2023AH51997)。

摘　　要：目的:探讨白细胞介素-34(IL-34)介导的核因子кB(NF-кB)对根尖牙乳头干细胞(SCAPs)成牙/成骨定向分化调节作用。方法:使用酶消化法从小鼠根尖牙乳头组织传代培养SCAPs;流式细胞术鉴定SCAPs中CD44、CD90、CD45及集落刺激因子1受体(CSF-1R)的表达;实验分为4组:空白对照组、IL-34组(100 ng/mL IL-34)、CSF-1R组(100 ng/mL IL-34+25 ng/mL anti-CSF-1R)、NF-кB组(100 ng/mL IL-34+1umol/L BMS-345541)。通过蛋白质印迹法(Western blot)分析各组30、60、120 min时SCAPs胞浆中NF-кB关键蛋白p-IкBα、IкBα、p-P65及P65蛋白的表达情况;矿化诱导4组SCAPs 3、7、14 d时,茜素红染色和半定量分析比较各组细胞的矿化结节形成能力;通过RT-qPCR检测各组SCAPs中成骨相关基因Runt相关转录因子-2(RUNX2)、牙本质涎磷蛋白(DSPP)、骨钙素(OCN)mRNA水平。结果:流式细胞术显示,SCAPs中CD44(90.4%)、CD90(93.5%)阳性表达,造血干细胞CD45(3.1%)阴性表达,SCAPs表达CSF-1R(23.2%)阳性。与对照组比较,IL-34组SCAPs胞浆蛋白内p-IкBα、p-P65表达水平上调,矿化诱导3、7、14 d矿化结节形成量增多,细胞中RUNX2、DSPP、OCN mRNA相对表达量升高,差异均有统计学意义(P<0.05)。与IL-34组比较,CSF-1R组和NF-кB组SCAPs胞浆蛋白内p-IкBα、p-P65表达水平下调(P<0.05),矿化诱导3、7、14 d矿化结节形成量减少,细胞中RUNX2、DSPP、OCN mRNA相对表达量降低,差异均有统计学意义(P<0.05)。结论:IL-34可通过IL-34/CSF-1R调节SCAPs中NF-кB信号通路,并通过IL-34/CSF-1R/NF-кB信号调节SCAPs的成牙/成骨定向分化能力。Objective:To investigate the regulatory role of nuclear factorкB(NF-кB)mediated by interleukin-34(IL-34)on the odontogenic/osteogenic differentiation of stem cells from the apical papilla(SCAPs).Methods:Enzymatic digestion method was used to subculture SCAPs from mouse apical papilla tissue.Flow cytometry was used to assess the expression of CD44,CD90,CD45,and colony-stimulating factor 1 receptor(CSF-1R)on the surface of SCAPs.The experiment was carried out in four groups:control group,IL-34 group(100ng/ml IL-34),CSF-1R group(100ng/mL IL-34+25ng/ml anti-CSF-1R),and NF-кB group(100ng/ml IL-34+1μmol/L BMS-345541).Western blot analysis was conducted at 30,60,and 120 minutes to examine the expression levels of NF-кB key proteins(p-IкBα,IкBα,p-P65,and P65)in the cytoplasm of SCAPs.Mineralization induction was performed on the four groups of SCAPs for 3,7,and 14 days,and Alizarin Red staining and semi-quantitative analysis were used to compare the mineralization nodule formation capacity of the cells.RT-qPCR was used to measure the mRNA levels of the osteogenic markers Runt-related transcription factor-2(RUNX2),dentin sialophosphoprotein(DSPP),and osteocalcin(OCN)in SCAPs of each group.Results:Flow cytometry analysis showed that in SCAPs expressed CD44(90.4%)and CD90(93.5%)were positive and were negative for hematopoietic stem cell marker CD45(3.1%).SCAPs expressed CSF-1R was positive(23.2%).Compared with the control group,SCAPs in the IL-34 group demonstrated significant upregulation of p-IкBαand p-P65 expression(P<0.05),increased mineralization nodule formation on the 3rd,7th,and 14th day,and a significant elevation in relative mRNA expression levels of RUNX2,DSPP,and OCN(P<0.05).Compared with the IL-34 group,the CSF-1R group and NF-кB group showed decreased expression of p-IкBαand p-P65 in the cytoplasm of SCAPs(P<0.05),reduced mineralization nodule formation on the 3rd,7th,and 14th day,and a significant decrease in relative mRNA expression levels of RUNX2,DSPP,and OCN(P<0.05).Conclusion:IL-34 can regu

关 键 词：根尖牙乳头干细胞 IL-34 定向分化 NF-кB信号通路 

分 类 号：R780.2[医药卫生—口腔医学]