ERK/MAPK信号通路在牙周膜干细胞成骨分化过程中的时空效应性变化

The spatial-temporal regulation of ERK/MAPK signaling pathway during the osteogenic differentiation of periodontal ligament stem cells

目的探讨牙周膜干细胞成骨分化进程中ERK/MAPK信号通路表达水平的变化,以及它对牙周膜干细胞成骨分化的调控作用。方法通过单克隆法获得人源性的牙周膜干细胞,分别用DMEM培养基、成骨诱导培养液处理牙周膜干细胞7、14、21d后收集细胞,提取总蛋白,通过Western Blot的方法检测ERK/MAPK通路的变化;分别用DMEM培养基、成骨诱导液、成骨诱导液+ERK抑制剂U0126处理牙周膜干细胞21d,通过茜素红染色的方法检测PDLSCs的成骨分化能力的改变;并通过分光光度计对茜素红染色结果进行定量分析。结果 DMEM组和成骨诱导组中,牙周膜干细胞胞内磷酸化ERK(P-ERK)的表达水平呈现先上升后下降的趋势,在诱导第14d达到峰值,在21d表达水平下降。在诱导第7d和第14d,成骨诱导组牙周膜干细胞胞内P-ERK/ERK的表达水平比值均高于DMEM组,具有显著统计学差异(P<0.05)。在第21d时,DMEM组牙周膜干细胞胞内P-ERK/ERK的表达水平比值略高于成骨诱导组,但无统计学差异(P>0.05)。ERK的抑制剂U0126的加入抑制牙周膜干细胞的成骨向分化过程中矿化结节的产生。结论 ERK/MAPK信号通路参与牙周膜干细胞的成骨向分化调控,它在牙周膜干细胞成骨分化过程中的表达随着时间的发展呈非线性变化。

Objective To investigate the expression of ERK/MAPK signaling pathway in the osteogenic differentiation of periodontal ligament stem cells(PDLSCs). Methods Human PDLSCs were acquired by clonal culture of healthy donor-derived molar. The human PDLSCs were treated with either DMEM or osteogenic medium, and 7, 14, 21 days later, the cytoplasmic protein was collected for Western Blot to examine the change of ERK/MAPK signaling pathway. Additionally, the osteogenic phenotype of human PDLSCs was determined and quantified by alizarin red staining after 21 days of treatment of DMEM or osteogenic medium in the presence or absence of U0126. Results The phosphorylation of ERK increased and peaked at 14 th day after osteogenic induction and dropped later in both DMEM group and osteogenic medium group. At 7 th and 14 th day after osteogenic induction, the phosphorylation level of ERK/MAPK was significantly higher in osteogenic medium group than that in DMEM group(P<0.05). At 21 st day after osteogenic induction, the phosphorylation level in DMEM group was higher than that in osteogenic medium group, but without significance(P>0.05). The addition of ERK inhibitor U0126 undermined the mineralization process and osteogenic differentiation of human PDLSCs. Conclusion ERK/MAPK signaling pathway was involved in the osteogenic differentiation of human PDLSCs, and the spatial-temporal change of ERK/MAPK activation was non-linear during osteogenic induction process.