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目的 :探讨转化生长因子β3(transforming growth factorβ3,TGF-β3)对成骨细胞内炎性细胞因子IL-6表达的影响,及其发挥抗炎作用的机制。方法:20μg/m L牙髓卟啉单胞菌脂多糖(lipopolysaccharide of Porphyromonas endodontalis,P.e-LPS)刺激人成骨肉瘤细胞系MG63,构建成骨细胞炎症模型。取不同浓度(5~20 ng/m L)的人重组蛋白生长因子TGF-β3和TGF-β1,分别与20μg/m L P.e-LPS共同作用于MG63细胞24 h后,利用实时荧光定量PCR检测细胞内IL-6 m RNA的表达,ELISA法检测培养液上清中IL-6的表达水平。以10 ng/m L TGF-β3预处理细胞30 min后,再与20μg/m L P.e-LPS共同作用20 min,Western印迹法检测细胞内ERK1/2蛋白磷酸化的表达。采用SPSS13.0软件包对数据进行统计学分析。结果:实时荧光定量PCR结果显示,单独20μg/m L P.e-LPS作用下,MG63细胞内IL-6的表达显著增高(P<0.01);TGF-β1在低浓度条件下(5~10 ng/m L)对IL-6的表达无显著作用,仅在20 ng/m L时可显著抑制IL-6的表达(P<0.05)。不同浓度的TGF-β3与P.e-LPS共同作用均可显著抑制IL-6的表达(P<0.01)。ELISA结果显示,10~20 ng/m L TGF-β3可在蛋白水平上对IL-6有显著抑制作用(P<0.05)。单独P.e-LPS作用时,可使MG63细胞内ERK1/2蛋白的磷酸化水平升高(P<0.01);而当TGF-β3与P.e-LPS共同作用时,ERK1/2的磷酸化被抑制(P<0.05)。结论:相同浓度条件下,TGF-β3比TGF-β1对成骨细胞炎症的抑制作用更为显著,ERK1/2信号机制参与了TGF-β3的抗炎过程。
Objective: To investigate the effect of transforming growth factor β3 (TGF-β3) on the expression of inflammatory cytokines IL-6 in osteoblasts and its anti-inflammatory mechanism. METHODS: Human osteosarcoma cell line MG63 was stimulated with 20 μg / mL lipopolysaccharide of Porphyromonas endodontalis (P.e-LPS) to establish an osteoblast inflammatory model. The recombinant human TGF-β3 and TGF-β1 with different concentration (5 ~ 20 ng / mL) were incubated with MG63 cells with 20 μg / mL Pe-LPS for 24 h. Real-time PCR The expression of IL-6 mRNA was detected by ELISA. The expression of IL-6 in the culture supernatant was detected by ELISA. The cells were pretreated with 10 ng / mL TGF-β3 for 30 min and then treated with 20 μg / mL P.e-LPS for 20 min. The phosphorylation of ERK1 / 2 protein was detected by Western blotting. SPSS13.0 software package for statistical analysis of the data. Results: The results of real-time PCR showed that IL-6 expression was significantly increased in MG63 cells treated with 20μg / mL Pe-LPS alone (P <0.01) m L) had no significant effect on the expression of IL-6. IL-6 expression was significantly inhibited only at 20 ng / mL (P <0.05). Different concentrations of TGF-β3 and P.e-LPS can significantly inhibit the expression of IL-6 (P <0.01). The results of ELISA showed that 10 ~ 20 ng / m L TGF-β3 could significantly inhibit IL-6 at the protein level (P <0.05). Phosphorylation of ERK1 / 2 in MG63 cells was increased by Pe-LPS alone (P <0.01), whereas phosphorylation of ERK1 / 2 was inhibited when TGF-β3 was combined with Pe-LPS (P < P <0.05). CONCLUSION: TGF-β3 inhibits osteoblast inflammation more than TGF-β1 at the same concentration. ERK1 / 2 signaling is involved in the anti-inflammatory process of TGF-β3.