白藜芦醇在人牙髓干细胞成牙本质向分化中的调控机制研究

Regulation mechanism of resveratrol on odontogenic differentiation of human dental pulp stem cells

目的:探讨白藜芦醇是否通过上调沉默信息调节因子1(silent information regulator 1,SIRT1)的表达并激活β-catenin信号通路,从而促进人牙髓干细胞(DPSCs)成牙本质向分化。方法:不同浓度白藜芦醇(0、10、15、20、50μmol/L)作用于DPSCs 7天和14天后,利用CCK-8法检测细胞增殖活性。在15μmol/L白藜芦醇作用下,成牙本质向分化诱导培养DPSCs 7天后,检测碱性磷酸酶(ALP)活性;采用实时定量反转录PCR(qRT-PCR)检测Runt相关转录因子2(Runx2)、牙本质涎磷蛋白(DSPP)和牙本质基质蛋白1(DMP-1)的mRNA表达情况;采用蛋白质免疫印迹法(Western blot)检测DPSCs分化诱导不同时间(0、3、5、7、14天)后SIRT1的蛋白表达情况。15μmol/L白藜芦醇作用下分化诱导培养DPSCs 7天后,检测SIRT1和活化β连环蛋白(β-catenin)表达水平。采用GraphPad Prism 9软件包对数据进行统计学分析。结果:15μmol/L白藜芦醇对DPSCs 7天和14天的增殖活性无明显影响;15μmol/L白藜芦醇促进DPSCs的成牙本质向分化,上调Runx2、DSPP和DMP-1的mRNA表达。DPSCs分化诱导7天时,SIRT1蛋白表达量最大;15μmol/L白藜芦醇作用下分化诱导DPSCs 7天时,SIRT1及活化β-catenin蛋白表达显著增加。结论:白藜芦醇通过上调SIRT1蛋白表达并激活β-catenin信号通路,促进人DPSCs成牙本质向分化。

PURPOSE:To investigate whether resveratrol promotes odontogenic differentiation of human dental pulp stem cells(DPSCs)by up-regulating the expression of silent information regulator 1(SIRT1)and activatingβ-catenin signaling pathway.METHODS:Different concentrations of resveratrol(0,10,15,20 and 50μmol/L)were used to treat DPSCs for 7 days and 14 days,and cell proliferative activity was detected by CCK-8.After odontogenic differentiation induced by 15μmol/L resveratrol for 7 days,alkaline phosphatase(ALP)staining was performed and real-time quantitative reverse transcription PCR(qRT-PCR)was used to detect the mRNA expression of Runt-related transcription factor 2(Runx2),dentin sialophosphoprotein(DSPP)and dentin matrix protein-1(DMP-1)in DPSCs.Western blot was used to detect the expression of SIRT1 in DPSCs on a specific day(0,3rd,5th,7th and 14th)after differentiation induction.Western blot was also used to detect the expression of SIRT1 and activatedβ-catenin during odontogenic differentiation of DPSCs treated by 15μmol/L resveratrol for 7 days.The experimental data was analyzed with GraphPad Prism 9 software package.RESULTS:15μmol/L resveratrol had no significant effect on proliferation of DPSCs on the 7th and 14th day;15μmol/L resveratrol promoted odontogenic differentiation of DPSCs and up-regulated mRNA expression of RUNX2,DSPP,and DMP-1 in DPSCs;the expression of SIRT1 was the highest on the 7th day during odontogenic differentiation induction.Resveratrol resulted in the increasing protein expressions of SIRT1 and activatedβ-catenin when DPSCs was induced to odontogenic differentiation for 7 days.CONCLUSIONS:Resveratrol promotes odontogenic differentiation of human DPSCs by up-regulating the expression of SIRT1 protein and activatingβ-catenin signaling pathway.