葛根素通过Wnt/β-catenin信号通路对牙髓干细胞的增殖及成骨向分化的作用机制研究

Effect of puerarin on the proliferation and osteogenic differentiation of dental pulp stem cells through Wnt/β-catenin signaling pathway

目的:探讨葛根素对牙髓干细胞体外增殖及成骨向分化的影响及其可能作用机制。方法:分离人牙髓干细胞(human dental pulp stem cells,h DPSCs)并培养鉴定,使用不含葛根素及含不同浓度葛根素(0.01、0.1、1、10μmol/L)的培养液干预,CCK-8法检测细胞增殖情况,筛选出最佳作用浓度;将细胞分为阴性对照组、阳性对照组及实验组,茜素红染色观察并分析各组h DPSCs矿化沉积情况,比色分析法检测各组细胞碱性磷酸酶(alkaline phosphatase,ALP)活性以评估葛根素对h DPSCs成骨向分化的影响;免疫荧光染色检测β-catenin细胞内表达情况;Western blot技术检测通路相关蛋白和细胞周期素D1(Cyclin D1)、Runt相关转录因子2(runt-related transcription factor 2,RUNX2)、骨钙素(osteocalcin,OCN)的蛋白表达水平。结果:经成骨化诱导及成脂化诱导检测,体外分离并培养、传代细胞为h DPSCs,具有成骨向分化潜能,可用于后续试验;CCK8检测结果显示,与不含葛根素组比较,0.01、0.1、1、10μmol/L葛根素组细胞吸光度值均升高,且1μmol/L葛根素作用下的细胞吸光度值最高(P<0.05);与阴性对照组比较,阳性对照组及实验组h DPSCs矿化沉积明显,矿化结节量、ALP活性升高(P<0.05),β-catenin在细胞质中强烈表达,h DPSCs中Wnt1、β-catenin、Cyclin D1、RUNX2、OCN蛋白表达水平升高(P<0.05),GSK-3β、p-GSK-3β蛋白表达水平降低(P<0.05);与阳性对照组比较,实验组细胞矿化结节量及ALP活性升高,h DPSCs中除p-GSK-3β蛋白表达水平降低外,Wnt1、β-catenin、Cyclin D1、RUNX2、OCN蛋白表达升高(P<0.05)。结论:葛根素可促进h DPSCs增殖及成骨向分化,其作用发挥机制可能与Wnt/β-catenin信号通路的激活有关。

Objective:To investigate the effect of Puerarin on the proliferation and osteogenic differentiation of dental pulp stem cells(DPSCs)in vitro and its possible mechanism.Methods:Human dental pulp stem cells(hDPSCs)were isolated,cultured and identified.Puerarin free and puerarin containing(0,0.01,0.1,1,10μmol/L)medium were introduced,the cell proliferation was detected by CCK-8 method,and the best concentration was selected thereof.Cells were divided into the negative control group,the positive control group,and the experimental group.Alizarin red staining was used to observe and analyze the mineralization and calcium deposition of hDPSCs in each group.Alkaline phosphatase(ALP)activity was detected by colorimetry to evaluate the effect of puerarin on the osteogenic differentiation of hDPSCs.The intracellular expression ofβ-catenin was detected by immunofluorescence staining.Protein expression levels of the signaling pathway related factors,runt-related transcription factors(RUNX2)and osteocalcin(OCN)were detected by western blot.Results:After ossification induction and adipogenesis induction detection,hDPSCs were isolated,cultured and passed in vitro with osteogenic differentiation potential,which could be used for subsequent tests.CCK8 showed that compared with 0μmol/L puerarin group,the absorbance value of 0.01,0.1,1 and 10μmol/L puerarin group was significantly increased,and the absorbance value of 1μmol/L puerarin group was the highest(P<0.05).The optimal concentration of puerarin promoting the proliferation of hDPSCs was 1μmol/L.Compared with cells in the negative control group,hDPSCs in the positive control group and the experimental group showed obvious mineralization deposition,and the number of mineralization nodules and ALP activity were significantly increased(P<0.05).β-catenin was strongly expressed in the cytoplasm.The protein expression levels of Wnt1,β-catenin,CyclinD1,RUNX2 and OCN in hDPSCs were significantly increased(P<0.05),while the protein expression levels of GSK-3βand p-GSK-3βwere significantly decreased(P<0.05).Compared with that in the positive control group,the number of cell mineralization nodules and ALP activity in the experimental group were significantly increased,the protein expression levels of Wnt1,β-catenin,CyclinD1,RUNX2 and OCN in hDPSCs were significantly increased,and the protein expression level of p-GSK-3βwas significantly decreased(P<0.05).Conclusion:Puerarin can promote the proliferation and osteogenic differentiation of hDPSCs,which may be related to Wnt/β-catenin signaling pathway.