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目的探讨在人骨髓间充质干细胞(hMSC)、小鼠前成骨细胞(MC3T3)和成牙本质细胞(MDPC-23)中,牙本质基质蛋白1(DMP1)是否通过激活丝裂原活化蛋白激酶(MAPK)-细胞外调节蛋白激酶(ERK)信号通路发挥作用。方法 Western blot检测不同时间点rDMP1C和rDMP1F蛋白处理hMSC、MC3T3-E1和MDPC-23后,对MAPK-ERK的激活情况;并检测使用MAPK抑制剂后,ERK的激活是否被抑制;细胞免疫荧光技术观察MAPK抑制剂抑制激活的ERK从胞浆向胞核的转位情况。Western blot检测siRNA沉默Ras基因后,对rDMP1引起MAPK-ERK信号通路激活的影响。结果三种细胞中,rDMP1F和rDMP1C在5 min~3 h均可以激活MAPK-ERK通路,而总ERK在各时间点均无显著变化;rDMP1F激活信号通路的持续时间均明显长于rDMP1C;MAPK抑制剂处理组的p-ERK条带与rDMP1F/C处理组的p-ERK条带差别明显。hMSC中,rDMP1F激活MAPK信号通路持续时间要长于其在MC3T3和MDPC-23中的作用。细胞免疫荧光检测发现,MAPK抑制剂组可以阻断ERK向细胞核内的转位。MC3T3和MDPC-23在siRNA沉默Ras基因后,ERK的磷酸化水平与rDMP1F单独处理组相比显著降低。结论 rDMP1C和rDMP1F都通过Ras激活MAPK-ERK信号通路,而rDMP1F比rDMP1C具有更强的信号功能。
Objective To investigate whether dentin matrix protein 1 (DMP1) activates mitogen-activated protein in human bone marrow mesenchymal stem cells (hMSCs), mouse pre-osteoblasts (MC3T3) and odontoblast cells Kinase (MAPK) - extracellular regulatory protein kinase (ERK) signaling pathway. Methods Western blot was used to detect the activation of MAPK-ERK after treated with rDMP1C and rDMP1F at different time points. The activation of MAPK-ERK was detected by using MAPK inhibitor. The immunofluorescence technique To observe the MAPK inhibitor inhibits the activation of ERK translocation from the cytoplasm to the nucleus. Western blot was used to detect the effect of rDMP1 on the activation of MAPK-ERK signaling pathway after siRNA silencing Ras gene. Results In all three cells, rDMP1F and rDMP1C activated MAPK-ERK pathway at 5 min to 3 h, while total ERK did not change significantly at all time points. The duration of rDMP1F activation signal pathway was significantly longer than rDMP1C; MAPK inhibitor The difference between the p-ERK band in the treatment group and the p-ERK band in the rDMP1F / C treatment group was significant. In hMSCs, rDMP1F activated MAPK signaling for longer than its role in MC3T3 and MDPC-23. Cell immunofluorescence assay revealed that MAPK inhibitor group could block ERK translocation into the nucleus. After MC3T3 and MDPC-23 siRNA silencing the Ras gene, the level of phosphorylation of ERK was significantly lower than that of rDMP1F alone. Conclusion Both rDMP1C and rDMP1F activate MAPK-ERK signaling pathway through Ras, while rDMP1F has stronger signal function than rDMP1C.