Toll样受体2激动剂Pam3CSK4对人牙髓细胞表达细胞因子的影响

Effect of Toll-like receptor 2 agonist Pam3CSK4 on the expression of several cytokines in human dental pulp cells

目的研究Toll样受体2（TLR2）激动剂Pam3CSK4对体外培养人牙髓细胞（hDPC）表达细胞因子的影响。方法特异性配体Pam3CSK4体外活化hDPC表面TLR2，实时荧光定量聚合酶链反应（PCR）检测不同时间处理后hDPC内白细胞介素6（IL-6）、IL-8、IL-1β及半胱氨酸-X-半胱氨酸趋化因子配体10（CXCLl0）mRNA的表达水平；双抗体夹心ELISA法检测上述细胞上清液中IL-6、IL-8蛋白的表达水平；激光共聚焦观察TLR2-核因子κB（NF-κB）信号通路关键蛋白p65的胞内分布情况。结果1μg／mlPam3CSK4处理hDPC0、4、8、12h，荧光定量PCR结果显示，除IL-6mRNA表达水平最先于4h达峰值外（P=0．006），IL-8、IL-1β及CXCLl0mRNA表达水平均于4h开始升高．8h达峰值（P〈0．001）：双抗体夹心ELISA法显示，hDPC上清液中IL-6及IL-8的蛋白表达于4h开始升高．8～12h趋于平稳。激光共聚焦显微镜发现，表达绿色荧光的NF-κBp65蛋白主要位于对照组细胞质．经1μg／mlPam3CSK4刺激75min后转移至胞核。结论特异性配体Pam3CSK4通过结合hDPC表面TLR2启动细胞内NF-κB信号通路进而激活其转录作用．诱导hDPC表达多种细胞因子．从而参与牙髓炎的早期免疫调控。

Objective This study was to investigate the effect of Toll-like receptor 2 agonist Pam3CSK4 on the expression of several cytokines in human dental pulp cells （hDPCs）. Methods The mRNA expression of IL-6, IL-8, IL-1β and CXCL10, and the protein expression of IL-6, IL-8 in hDPCs with 1 μg/ml Pam3CSK4 at different time points were examined by real-time quantitative PCR and ELISA, respectively. Nuclear location of p65 was investigated by immunofluorescent staining in hDPCs. Results The mRNA expression of IL-6 firstly elevated at 4 h, whereas the expression of IL-8, IL-1β and CXCL10 peaked at 8 hours by the incubation with 1 μg/ml Pam3CSK4. IL-6 and IL-8 proteins were clearly expressed in hDPCs culture supernatant, which increased at 4 h and maintained stable at 8 h. Confocal laser scanning microscope showed the positive expression of p65 in nuclear with 1 μg/ml Pam3CSK4 for 75 min, whereas p65 was only observed in cytoplasm in untreated control group. Conclusions Activation of TLR2 by Pam3CSK4 induced the expression of several cytokines both at mRNA and protein levels at 4 - 8 h through promoting NF-KB activation in hDPCs, indicating they may be involved in immune regulation of early pulpitis.