TNF-α在慢性根尖周炎骨破坏中的机制探讨

Mechanism of TNF-α in bone defect of chronic apical periodontitis

目的探讨牙髓卟啉单胞菌（Porphyromonas endodontalis,P.e）脂多糖（lipopolysaccharide,LPS）是否诱导成骨细胞产生肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）及TNF-α是否通过核因子κB（nuclear factor-κB,NF-κB）信号通路上调巨噬细胞集落刺激因子（macrophage colony stimulating factor,M-CSF）的产生。方法以不同浓度P.e-LPS （0~50 mg/L）刺激MC3T3-El细胞和以10 mg/L P.e-LPS作用细胞不同时间（0~24 h）后,采用反转录-聚合酶链式反应（RT-PCR）检测TNF-α mRNA的表达;以不同浓度TNF-α（0~10 ng/L） 刺激MC3T3-El细胞后,采用RT-PCR和酶联免疫吸附试验（ELISA）检测M-CSF mRNA和蛋白的表达;再以ELISA方法检测BAY 11-7082对TNF-α 刺激MC3T3-El细胞后M-CSF蛋白表达的影响。采用SPSS 13.0软件包对结果进行单因素方差分析和Dunnett t检验。结果不同质量浓度的P.e-LPS（0~50 mg/L）刺激MC3T3-El细胞后,TNF-α mRNA的表达具有剂量依赖性;10 mg/L P.e-LPS作用MC3T3-El细胞6 h时,TNF-α mRNA 的表达量最大。随着作用时间的延长,TNF-α mRNA 的表达量逐渐下降;不同质量浓度TNF-α（0~10 ng/L）刺激MC3T3-El细胞后,M-CSF mRNA 和蛋白的表达具有剂量依赖性,蛋白表达量从（37±2） ng/L（空白对照组）增加到（301±8） ng/L（10 ng/L组）;10 mol/L BAY 11-7082预处理1 h可以降低TNF-α诱导成骨细胞M-CSF蛋白的表达水平,蛋白表达量从（253±14） ng/L（TNF-α组）下降到（154±2）ng/L（BAY＋TNF-α组）,而单独使用BAY 11-7082组与空白对照组相比差异无显著性。结论P.e-LPS诱导成骨细胞产生TNF-α,而TNF-α 上调M-CSF可能通过NF-κB信号通路,这意味着TNF-α可能在P.e-LPS致慢性根尖周炎骨破坏中对成骨细胞发挥自分泌作用。

PURPOSE： To investigate the effect of lipopolysaccharides（LPS） extracted from Porphyromonas endodontalis（P.e） on the expression of tumor necrosis factor-α（TNF-α ） mRNA in MC3T3-E1 cells and the role of NF-κB signaling on the expression of macrophage colony stimulating factor （M-CSF） induced by TNF-α in MC3T3-El cells. METHODS： MC3T3-E1 cells were treated with different concentrations of P.e-LPS（0-50 mg/L） and 10 mg/L P.e-LPS for different time （0-24 h）. The expression of TNF-α mRNA was detected by reverse transcription polymerase chain reaction（RT-PCR）. MC3T3-E1 cells were treated with different concentrations of TNF-α（0-10 ng/L） for 6 h. The expression of M-CSF mRNA and protein was detected by RT-PCR and enzyme-linked immunoadsordent assay（ELISA）.The expression of M-CSF protein was also detected in 10 ng/L TNF-α treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor . Statistical analysis was performed using Multi-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS： The level of TNF-α mRNA increased significantly after treatment with different concentrations of P.e-LPS（0-50 mg/L）,which indicated that P.e-LPS induced osteoblasts to express TNF-α mRNA in dose dependent manners. Maximal induction of TNF-α mRNA expression was seen in the MC3T3-E1 cells treated with 10 mg/L P.e-LPS for 6 h. After 6 h, the expression of TNF-α mRNA decreased gradually .The expression of M-CSF mRNA and protein was increased in a does- dependent manner by different concentrations of TNF-α treatment（0-10 ng/L）. The expression of M-CSF protein increased from （37±2） ng/L（control group） to （301±8） ng/L（10 ng/L group）.The protein of M-CSF decreased significantly after pretreatment with 10 μmol/L BAY 11-7082 for 1 h, and the expression of M-CSF proteins was reduced from （253±14） ng/L to （154±2） ng/L .BAY group had no significant difference from the control group. CONCLUSIONS： The expression of TNF-α mRNA was increased by P. endodontalis LPS treatment in osteoblast. TNF-α may induce the expression of M-CSF in MC3T3-E1 cells through the signaling of NF-κB. It suggests that TNF-α affect osteoblasts through autocrine way for bone destruction in chronic apical periodontitis induced by P.e-LPS.