变形链球菌对EAhy926细胞Toll样受体2和4及白细胞介素6和8表达的影响

Effects of Streptococcus Mutans on the Expression of Toll Like Receptor 2 and 4,Interleukin-6 and-8 in EAhy926 Cells

目的观察变形链球菌对EAhy926细胞Toll样受体2和4的表达及炎性细胞因子白细胞介素6和8分泌的影响,初步探讨Toll样受体表达与细胞因子产生之间的关系。方法变形链球菌作用于EAhy926细胞,RT-PCR法检测EAhy926细胞Toll样受体2和4及白细胞介素6和8的mRNA表达;流式细胞术检测EAhy926细胞表面Toll样受体2和4的表达;细胞生物活性法和ELISA分别检测EAhy926细胞白细胞介素6和8的分泌;抗体阻断实验观察Toll样受体2和4的表达与白细胞介素6和8产生间的关系。结果将变形链球菌作用于EAhy926细胞6 h,Toll样受体2和4的mRNA表达与加入的细菌量呈一定的剂量依赖关系,以细菌与细胞作用比例为100∶1时,Toll样受体2和4的mRNA表达量最高(P<0.05)。变形链球菌能诱导EAhy926细胞(100∶1)Toll样受体2和4的mRNA和蛋白水平表达增强,在6 h达到高峰,12 h后又逐渐下降(P<0.01);白细胞介素6和8的表达量也增加,在12 h达最大表达量(P<0.05)。经Toll样受体2和Toll样受体4抗体阻断后,变形链球菌诱导EAhy926细胞白细胞介素6和8的mRNA表达明显减少(P<0.01)。结论变形链球菌可上调EAhy926细胞Toll样受体2和4的表达,促进炎性细胞因子白细胞介素6和8的产生;白细胞介素6和8的产生与Toll样受体2和4的表达上调密切相关。

Aim To investigate the effects of streptococcus mutans on the expression of toll-like receptor 2（TLR2）,TLR4,interleukin-6（IL-6） and IL-8 in EAhy926 cells（the human endothelial hybridoma of human umbilical vein endothelial cells（HUVEC） and the human epithelial cell line A549,characterized by endothelial phenotype and biology）. Methods EAhy926 cells were treated with Streptococcus mutans.The mRNA expression of TLR2,TLR4,IL-6 and IL-8 in EAhy926 cells was detected by reverse transcription polymerase chain reaction（RT-PCR）.TLR2 and TLR4 were analyzed by flow cytometry.The production of IL-6 and IL-8 in the cultured supernatants was measured by biochemical method and ELISA respectively.TLR2 and TLR4 blocking assay was used to investigate the relationship between IL-6,IL-8 and TLR2,TLR4 mRNA expression. Results The expression of mRNA and protein for TLR2 and TLR4 in EAhy926 cells increased after stimulated by Streptococcus mutans,peaked the maximal level at 6 h（P〈0.05）,and then decreased.The expression of IL-6 and IL-8 mRNA was significantly induced when exposed to Streptococcus mutans,reaching the maximal level at 12 h,respectively（P〈0.05）.Meanwhile,Streptococcus mutans induced the production of IL-6 and IL-8 with peaking at 12 h（P〈0.01）.The mRNA expression of IL-6 and IL-8 in EAhy926 cells was significantly blocked by anti-human TLR2 and anti-TLR4. Conclusion Streptococcus mutans upregulated the expression of TLR2 and TLR4 and induced the production of inflammatory cytokines IL-6 and IL-8.The expression of TLR2 and TLR4 of EAhy926 cells may elicit a TLR2 and TLR4-mediated innate immune response and contribute to production of inflammatory cytokines IL-6 and IL-8.