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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-αm RNA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 m RNA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-αm RNA的表达具有剂量依赖性;10 mg/L P.eLPS作用MC3T3-El细胞6 h时,TNF-αm RNA的表达量最大。随着作用时间的延长,TNF-αm RNA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 m RNA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。
Objective: To investigate whether Porphyromonas endodontalis (Pe) lipopolysaccharide (LPS) can induce osteoclasts to produce tumor necrosis factor-α (TNF-α) and TNF-α Nuclear factor-κB (NF-κB) signaling pathway upregulates the production of macrophage colony stimulating factor (M-CSF). Methods: MC3T3-El cells were stimulated with different concentrations of Pe-LPS (0-50 mg / L) and treated with 10 mg / L Pe-LPS for different time (0-24 h). Reverse transcription-polymerase chain The expression of TNF-αm RNA was detected by RT-PCR. After stimulation of MC3T3-El cells with different concentrations of TNF-α (0-10 ng / L), RT-PCR and enzyme-linked immunosorbent assay M-CSF m RNA and protein were detected by ELISA. The effect of BAY 11-7082 on the expression of M-CSF protein in MC3T3-El cells stimulated by TNF-α was detected by ELISA. One-way ANOVA and Dunnett t-test were used to analyze the results using SPSS 13.0 software package. Results: The expression of TNF-αmRNA was dose-dependent when MC3T3-El cells were stimulated with different concentrations of Pe-LPS (0-50 mg / L). MC3T3-E1 cells were treated with 10 mg / L P.eLPS for 6 h , TNF-αm RNA expression was the largest. The expression of TNF-αmRNA gradually decreased with the prolongation of time, and the expression of m-CSF m RNA and protein was dose-dependent when MC3T3-El cells were treated with different concentrations of TNF-α (0-10 ng / L) (37 ± 2) ng / L (blank control group) to (301 ± 8) ng / L (10 ng / L group), and 10 mol / L BAY 11-7082 pretreatment for 1 h Decreased the expression of M-CSF protein in osteoblasts from (253 ± 14) ng / L (TNF-α group) to (154 ± 2) ng / L Group), while BAY 11-7082 alone group compared with the blank control group no significant difference. CONCLUSION: Pe-LPS induces osteoblasts to produce TNF-α, whereas upregulation of M-CSF by TNF-α may pass through the NF-κB signaling pathway, suggesting TNF-α may be involved in bone destruction caused by Pe-LPS-induced chronic apical periodontitis Osteoblasts play an autocrine effect.