中国实用口腔科杂志

中国实用口腔科杂志

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小檗碱对牙髓干细胞MAPK/ERK信号通路及成牙本质向分化的影响研究

毕磊，刘辉，武燃   

  1. 华北理工大学附属医院口腔科，河北 唐山 063000
  • 出版日期:2021-01-30 发布日期:2021-02-08
  • 基金资助:
    河北省2019年度医学科学研究课题计划（20190109）

  • Online:2021-01-30 Published:2021-02-08

摘要： 目的　探讨小檗碱对牙髓干细胞（dental pulp stem cells，DPSCs）成牙本质向分化及p38丝裂原活化蛋白激酶（MAPK）/细胞外调节蛋白激酶（ERK）信号通路的影响。方法　采用酶消化法提取DPSCs，将培养鉴定的DPSCs传代培养至第3 ~ 5代，用于以下实验。实验分为对照组（正常培养DPSCs）、矿化液组（加入矿化液）、小檗碱组（加入10 μmol/L小檗碱）、矿化液+MAPK抑制剂组（加入矿化液+10 μmol/L MAPK抑制剂SB203580）、小檗碱+MAPK抑制剂组（10 μmol/L小檗碱+10 μmol/L MAPK抑制剂SB203580）。CCK-8法检测各组培养1、7、10、14 d时的细胞增殖情况。各组处理14 d，茜素红染色观察细胞矿化情况；实时荧光定量PCR检测细胞碱性磷酸酶（ALP）、牙本质涎磷蛋白（DSPP）、骨桥蛋白（OPN） mRNA表达水平；蛋白免疫印迹检测细胞p38 MAPK、磷酸化p38 MAPK（p-p38 MAPK）、ERK、磷酸化ERK（p-ERK）蛋白表达水平。结果 5组细胞处理1、7、10、14 d，对照组、矿化液组、小檗碱组光密度（OD）值差异无统计学意义（P > 0.05），说明3组的细胞增殖能力相似；分别与对照组、矿化液组、小檗碱组相比，矿化液+MAPK抑制剂组和小檗碱+MAPK抑制剂组的OD值升高（均P < 0.05），说明其细胞增殖能力增强。与对照组相比，矿化液组和小檗碱组的DPSCs矿化结节形成能力增强，细胞中ALP、DSPP、OPN的mRNA表达水平以及p-p38 MAPK/p38 MAPK、p-ERK/ERK蛋白表达水平比值升高（P < 0.05）。分别与矿化液组、小檗碱组相比，矿化液+MAPK抑制剂组、小檗碱+MAPK抑制剂组的DPSCs矿化结节形成能力减弱，细胞中ALP、DSPP、OPN的mRNA表达水平以及p-p38 MAPK/p38 MAPK、p-ERK/ERK蛋白表达水平比值降低（P < 0.05）。结论 小檗碱可促进DPSCs成牙本质向分化，可能是通过激活p38 MAPK/ERK信号通路实现的。

关键词: 小檗碱, 牙髓干细胞, 成牙本质向分化, 丝裂原活化蛋白激酶/细胞外调节蛋白激酶信号通路

Abstract: Objective To investigate the effects of berberine on the odontogenic differentiation of dental pulp stem cells（DPSCs）and the signal pathway of p38 mitogen activated protein kinase（MAPK）/extracellular regulated protein kinase（ERK）. Methods The DPSCs were extracted by enzymatic digestion，and the DPSCs identified in the culture were subcultured to the 3rd to 5th generation for the following experiments. The experiment included：control group （normal culture of DPSCs），mineralized liquid group（addition of mineralized liquid），berberine group（10 μmol/L berberine），mineralized liquid + MAPK inhibitor group（mineralized liquid + 10 μmol/L SB203580），berberine + MAPK inhibitor group（10 μmol/L berberine + 10 μmol/L SB203580）. CCK8 was used to detect cell proliferation at 1，7，10，and 14 days of culture in each group. After 14 days of treatment，the cell mineralization was observed by staining with Alizarin Red；the mRNA levels of cellular alkaline phosphatase（ALP），dentin sialophosphoprotein（DSPP），and osteopontin（OPN）were detected by qRT-PCR；Western blot was used to detect the expression levels of cell p38 MAPK，p-p38 MAPK，ERK，p-ERK protein. Results There was no significant difference in OD among the control group，the mineralized liquid group，and the berberine group（P > 0.05）after the 5 groups were treated for 1，7，10，and 14 days，which indicated that the cell proliferation ability of the 3 groups was similar；compared with the control group，mineralized liquid group and berberine group respectively，the OD of the mineralized liquid + MAPK inhibitor group and the berberine + MAPK inhibitor group increased（P < 0.05），which indicated that the cell proliferation ability was increased. Compared with the control group，the ability of dental pulp stem cells to mineralize nodules in the mineralized liquid group and berberine group was enhanced，and the mRNA levels of ALP，DSPP，OPN，and the protein expression level ratio of p-p38 MAPK/p38 MAPK and p-ERK/ERK in the cells increased（P < 0.05 ）. Compared with the mineralized fluid group and the berberine group，respectively，the DPSCs of mineralized fluid + MAPK inhibitor group and the berberine + MAPK inhibitor group had weaker ability to mineralize nodules，and the mRNA expression level of ALP，DSPP and OPN，and the protein expression level ratio of p-p38 MAPK/p38 MAPK and p-ERK/ERK decreased（P < 0.05）. Conclusion Berberine can promote the odontogenic differentiation of dental pulp stem cells，which may be achieved by activating p38 MAPK/ERK signal pathway.

Key words: berberine, dental pulp stem cells, odontogenic differentiation, mitogen activated protein kinase/extracellular regulated protein kinase signal pathway