TNF-α对人牙囊细胞表达单核细胞趋化蛋白-1的影响及其信号传导通路

Effects of TNF-α on the expression of monocyte chemoattractant protein-1 and the corresponding signal transduction pathway in dental follicle cells

目的研究不同浓度肿瘤坏死因子α(TNF-α)对体外培养人牙囊细胞表达单核细胞趋化蛋白-1(MCP-1)的影响及其信号传导通路。方法取第5代人牙囊细胞,分别与浓度为0(对照组)5、1、02、55、0、100ng/ml的TNF-α共孵育6h,夹心ELISA法检测上清液中MCP-1的含量,RT-PCR法检测MCP-1 mRNA表达的变化。另取第5代人牙囊细胞,分别加入25μmol/L SB203580、50μmol/L PD98059、15μmol/L SP600125,以阻断p38丝裂原活化蛋白激酶(p38MARK)、细胞外信号调节激酶(ERK)J、un氨基端激酶(JNK),培养30min后加入10ng/ml TNF-α,孵育6h后,RT-PCR检测MCP-1 mRNA含量。结果 ELISA结果显示,与对照组比较,TNF-α浓度为10～100ng/ml时,可显著增强MCP-1的分泌(P<0.05);RT-PCR结果显示,与对照组比较,TNF-α浓度为10～50ng/ml时,MCP-1表达增强,且此作用可被阻滞剂SP600125阻断。结论 TNF-α可增强MCP-1的基因表达、蛋白合成和分泌,此作用通过JNK信号转导途径实现。

Objective To study the effect of different concentration of tumor necrosis factor-α（TNF-α） on the expression of monocyte chemoattractant protein-1（MCP-1） and the corresponding signal transduction pathway in human dental follicle cells.Methods The 5th passage of human dental follicle cells were co-incubated with 0（control group）,5,10,25,50 and 100 ng/ml TNF-α,respectively,for 6 hours.The contents of MCP-1 in the supernatant were measured by using sandwich ELISA,and the expression of MCP-1 mRNA was determined by reverses transcription polymerase chain reaction（RT-PCR）.Furthermore,to determine the corresponding signal transduction pathway,the 5th passage of human dental follicle cells were incubated with 25 μmol/L SB203580 to inhibit p38 mitogen-activated protein kinase（p38MARK）,with 50 μmol/L PD98059 to inhibit extracellular signal-regulated kinases（ERK）,and with 15 μmol/L SP600125 to inhibit c-Jun N-terminal kinases（JNK） for 30min,then incubated with TNF-α（10ng/ml） for 6h.MCP-1 mRNA was detected by RT-PCR.Results The results of ELISA revealed that 10-100 ng/ml of TNF-α enhanced MCP-1 secretion（P〈0.05） compared to that in human dental follicle cells without TNF-α treatment.Cells treated with 10-50 ng/ml of TNF-α showed a significant increase of MCP-1 mRNA expression（P〈0.05）,and the action was inhibited by SP600125,which was the special inhibitor of c-Jun N-terminal kinase（JNK）.Conclusion TNF-α may enhance MCP-1 gene expression and secretion in human dental follicle cells,and the activation of JNK signal transduction pathway is required in this process.