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中华口腔医学研究杂志(电子版) ›› 2016, Vol. 10 ›› Issue (03) : 166 -171. doi: 10.3877/cma.j.issn.1674-1366.2016.03.003

所属专题: 文献

基础研究

促红细胞生成素对人牙髓细胞迁移能力的作用
曾俊瑜1, 龚启梅1, 凌均棨1,()   
  1. 1. 510055 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室
  • 收稿日期:2016-02-27 出版日期:2016-06-01
  • 通信作者: 凌均棨
  • 基金资助:
    中山大学青年教师培育计划(12ykpy65)

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

Junyu Zeng1, Qimei Gong1, Junqi Ling1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2016-02-27 Published:2016-06-01
  • Corresponding author: Junqi Ling
  • About author:
    Corresponding author: Ling Junqi, Email:
引用本文:

曾俊瑜, 龚启梅, 凌均棨. 促红细胞生成素对人牙髓细胞迁移能力的作用[J]. 中华口腔医学研究杂志(电子版), 2016, 10(03): 166-171.

Junyu Zeng, Qimei Gong, Junqi Ling. The study on the role of erythropoietin in migration of human dental pulp cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2016, 10(03): 166-171.

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mRNA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 mRNA的表达(tCXCR4= 5.727,PCXCR4= 0.005;tSDF-1= 3.412,PSDF-1= 0.027);与对照组相比,EPO显著促进hDPC的迁移能力(F= 207.775,P10 U/ml= 0.000,P20 U/ml= 0.000,P40 U/ml= 0.000);EPO可升高MAPK信号通路中关键蛋白ERK1/2(t15 min= 6.554,P15 min= 0.000;t30 min= 17.305,P30 min= 0.000;t60 min= 8.913,P60 min= 0.000;t120 min=-5.896,P120 min= 0.934)和p38的磷酸化程度(t15 min= 4.396,P15 min= 0.004;t30 min= 6.447,P30 min= 0.000;t60 min= 34.676,P60 min= 0.000;t120 min= 4.689,P120 min= 0.003);MAPK信号通路抑制剂U0126、SB203580可抑制EPO诱导的hDPC迁移(tEPO-U0126= 2.422,PEPO- U0126= 0.025;tEPO-SB203580= 3.837,PEPO-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 mRNA levels of chemotaxis factors were measured by quantitative polymerase chain reaction (qPCR) . Transwell migration assay was conducted to estimate the effect of EPO on the migration of hDPCs. The phosphorylated levels of ERK, p38 and JNK in hDPCs after stimulation of EPO within 120min were determined by western blot. Wound-healing migration assay was conducted to observe the effect of MAPK inhibitors on migration induced by EPO in hDPCs.

Results

The mRNA expression of CXCR4 and SDF-1 were enhanced by EPO (tCXCR4= 5.727, PCXCR4= 0.005; tSDF-1= 3.412, PSDF-1= 0.027) . The migration ability of hDPCs was improved under EPO in comparison to control group (F= 207.775, P10 U/ml= 0.000, P20 U/ml= 0.000, P40 U/ml= 0.000) . Both p-ERK (t15 min= 6.554, P15 min= 0.000; t30 min= 17.305, P30 min= 0.000; t60 min= 8.913, P60 min= 0.000; t120 min=-5.896, P120 min= 0.934) and p-p38 (t15 min= 4.396, P15 min= 0.004; t30 min= 6.447, P30 min= 0.000; t60 min= 34.676, P60 min= 0.000; t120 min= 4.689, P120 min= 0.003) were upregulated within 120 min after EPO treatment. Wound-healing migration assay showed that the migration ability of hDPCs 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 mRNA expression of chemotaxis factors CXCR4 and SDF-1, and enhanced hDPCs migration through activating MAPK pathway.

表1 PCR引物序列
图1 实时荧光定量PCR检测MCP-1、FGF-2、CXCR4、SDF-1 mRNA表达水平
图2 Transwell小室实验检测EPO对人牙髓细胞迁移能力的影响
图3 Western blot检测MAPK信号通路ERK、p38、JNK蛋白磷酸化程度的变化
图4 细胞划痕实验检测MAPK通路抑制剂对EPO诱导人牙髓细胞迁移的影响
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