促红细胞生成素对人牙髓细胞迁移能力的作用

The study on the role of erythropoietin in migration of human dental pulp cells

目的研究促红细胞生成素(EPO)对人牙髓细胞(hDPC)迁移能力的影响,并初步探讨相关分子机制。方法实时荧光定量聚合链反应(PCR)检测EPO对hDPC表达趋化因子m RNA的影响;Transwell实验观察不同浓度的EPO对hDPC迁移能力的影响;Western blot检测不同时间点hDPC中p38、ERK1/2、JNK磷酸化水平的变化;细胞划痕实验观察丝裂原活化蛋白激酶(MAPK)信号通路抑制剂对EPO诱导hDPC迁移的影响。结果 EPO上调趋化因子CXCR4、SDF-1 m RNA的表达(t_(CXCR4)=5.727,P_(CXCR4)=0.005;t_(SDF-1)=3.412,P_(SDF-1)=0.027);与对照组相比,EPO显著促进hDPC的迁移能力(F=207.775,P_(10U/ml)=0.000,P_(20U/ml)=0.000,P_(40U/ml)=0.000);EPO可升高MAPK信号通路中关键蛋白ERK1/2(t_(15min)=6.554,P_(15min)=0.000;t_(30 min)=17.305,P_(30 min)=0.000;t_(60 min)=8.913,P_(60 min)=0.000;t_(120 min)=-5.896,P_(120 min)=0.934)和p38的磷酸化程度(t_(15min)=4.396,P_(15min)=0.004;t_(30 min)=6.447,P_(30 min)=0.000;t_(60 min)=34.676,P_(60 min)=0.000;t_(120 min)=4.689,P_(120 min)=0.003);MAPK信号通路抑制剂U0126、SB203580可抑制EPO诱导的hDPC迁移(t_(EPO-U0126)=2.422,P_(EPO-U0126)=0.025;t_(EPO-SB203580)=3.837,P_(EPO-SB203580)=0.001)。结论 EPO上调趋化因子CXCR4和SDF-1 mRNA的表达,通过激活MAPK信号通路,促进hDPC迁移。

Objective To investigate the effect of erythropoietin on migration of human dental pulp cells and preliminarily explore the mechanisms. Methods The m RNA levels of chemotaxis factors were measured by quantitative polymerase chain reaction（q PCR）. Transwell migration assay was conducted to estimate the effect of EPO on the migration of h DPCs. The phosphorylated levels of ERK,p38 and JNK in h DPCs after stimulation of EPO within 120 min were determined by western blot. Wound-healing migration assay was conducted to observe the effect of MAPK inhibitors on migration induced by EPO in h DPCs.Results The m RNA expression of CXCR4 and SDF-1 were enhanced by EPOtCXCR4=5.727,PCXCR4=0.005;tSDF-1= 3.412,PSDF-1= 0.027）. The migration ability of h DPCs was improved under EPO in comparison to control group（F = 207.775,P10U/ml= 0.000,P20U/ml= 0.000,P40U/ml= 0.000）. Both p-ERK（t15min= 6.554,P15min=0.000;t30 min= 17.305,P30 min= 0.000;t60 min= 8.913,P60 min= 0.000;t120 min=-5.896,P120 min= 0.934and p-p38t15min= 4.396,P15min= 0.004;t30 min= 6.447,P30 min= 0.000;t60 min= 34.676,P60 min= 0.000;t120 min= 4.689,P120 min=0.003were upregulated within 120 min after EPO treatment. Wound-healing migration assay showed that the migration ability of h DPCs was inhibited by U0126 and SB203580 pretreatment tEPO-U0126= 2.422,PEPO-U0126= 0.025;tEPO-SB203580= 3.837,PEPO-SB203580= 0.001）. Conclusion EPO upregulated m RNA expression of chemotaxis factors CXCR4 and SDF-1,and enhanced h DPCs migration through activating MAPK pathway.