重组人成纤维细胞生长因子21调节成牙骨质细胞矿化作用及其机制研究

Effect of recombinant human fibroblast growth factor 21 on the mineralization of cementoblasts and its related mechanism

目的探讨重组人成纤维细胞生长因子21(rhFGF21)对成牙骨质细胞增殖和矿化的作用及其机制。方法采用苏木精-伊红染色、免疫组织化学染色和免疫荧光法检测成纤维细胞生长因子21(FGF21)在大鼠牙周组织和成牙骨质细胞OCCM-30中的表达和分布;采用CCK-8法检测经不同浓度rhFGF21处理OCCM-30的增殖情况;采用碱性磷酸酶染色和茜素红染色分别检测OCCM-30矿化诱导3、7 d后的矿化状态;通过实时定量聚合酶链反应(PCR)及蛋白免疫印迹法(Western blot)检测成骨相关基因Runx2和Osterix的转录及蛋白表达情况;通过PCR阵列分析评估OCCM-30内转化生长因子β(TGFβ)/骨形态发生蛋白(BMP)信号通路基因的表达变化。结果FGF21在大鼠牙周组织和OCCM-30中存在表达;rhFGF21对OCCM-30的增殖能力无明显影响,但50 ng/mL rhFGF21能促进OCCM-30矿化能力增强(P<0.001);实时定量PCR结果显示Runx2和Osterix的转录水平在50 ng/mL rhFGF21矿化诱导3 d时上升,5 d时下降;Western blot结果显示,Runx2及Osterix的蛋白表达水平在矿化诱导过程中增强,rhFGF21上调细胞内Bmpr1b的表达。结论rhFGF21能够促进OCCM-30矿化,这与TGFβ/BMP信号通路的激活有关。

Objective To investigate the effect of recombinant human fibroblast growth factor 21(rhFGF21)on the proliferation and mineralization of cementoblasts and its mechanism.Methods Hematoxylin eosin,immunohistochemical staining,and immunofluorescence were used to detect the expression and distribution of fibroblast growth factor 21(FGF21)in rat periodontal tissues and cementoblasts(OCCM-30),separately.Cell Counting Kit-8 was used to detect the proliferation of OCCM-30 undertreatment with rhFGF21.Alkaline phosphatase staining and Alizarin Red staining were used to detect the mineralization state of OCCM-30 after 3 and 7 days of mineralization induction.The transcription and protein expression of the osteogenic-related genes Runx2 and Osterix were detected by real-time quantitative polymerase chain reaction(PCR)and Western blot analysis.The expression levels of genes of transforming growth factorβ(TGFβ)/bone morphogenetic protein(BMP)signaling pathway in OCCM-30 were detected through PCR array analysis.Results FGF21 was expressed in rat periodontal tissues and OCCM-30.Although rhFGF21 had no significant effect on the proliferation of OCCM-30,treatment with 50 ng/mL rhFGF21 could promote the mineralization of OCCM-30 cells after 7 days of mineralization induction.The transcriptional levels of Runx2 and Osterix increased significantly at 3 days of mineralization induction and decreased at 5 days of mineralization induction.Western blot analysis showed that the protein expression levels of Runx2 and Osterix increased during mineralization induction.rhFGF21 up-regulated Bmpr1b protein expression in cells.Con⁃clusion rhFGF21 can promote the mineralization ability of OCCM-30.This effect is related to the activation of the TGFβ/BMP signaling pathway.