瑞香素对B淋巴细胞TLR1～10mRNA及IL-12 mRNA表达的影响

Effects of Daphnetin on the mRNA Expression of Toll-like Receptors(TLRs) 1～10 and IL-12 of B lymphocytes

目的观察瑞香素对人外周血B淋巴细胞TLR1～10 mRNA及IL-12 mRNA表达的影响,研究瑞香素对B淋巴细胞作用的可能机制。方法尼龙毛柱分离法分离T、B淋巴细胞;MTT法探讨瑞香素作用B淋巴细胞的适宜浓度;采用实时荧光定量PCR(RT-PCR)检测瑞香素对B细胞TLR1～10 mRNA及IL-12 mRNA表达影响,同时检测用抗TLR4单抗封闭B细胞TLR4后TLR mRNA及IL-12 mRNA表达的变化。结果瑞香素浓度为6.25～25.00μg/ml时对B细胞增殖有明显增强作用(P<0.05),与SPA有较好协同作用,以浓度为12.50μg/ml瑞香素作用最强;瑞香素作用的未封闭组与封闭组的B细胞TLR4 mRNA、TLR9 mRNA、IL-12 mRNA表达均明显高于对照组(P<0.05);未封闭组B细胞TLR4 mR-NA、TLR9 mRNA、IL-12 mRNA表达量明显高于封闭组(P<0.05)。结论天然免疫途径中TLR4、TLR9参与瑞香素从基因转录水平增加IL-12的分泌,从而增强B淋巴细胞免疫功能。

Objective To study the mechanism of daphnetin regulating B lymphocytes in human peripheral blood by observing the effect of daphnetin on the expression of TLR1～10 mRNA,IL-12 mRNA.Methods B lymphocytes were separated by Nylon Fiber Column methods.The optimal concentration of daphnetin on the proliferation of B lymphocytes was evaluated by MTT assay.The effect of daphnetin on the mRNA expression of TLR1-10,IL-12 were determined by the real-time fluorescence quantitative polymerase chain reaction（RT-PCR）.Furthermore,the mRNA expression of TLRs and cytokine were tested again by RT-PCR after blocking with anti-TLR4.Results Daphnetin could significantly promote the proliferation of B lymphocytes at the concentration of 6.25,12.50,25.00 μg/ml（P﹤0.05）;Moreover,it（12.50,25.00 μg/ml） could notably promote the proliferation of B lymphocytes stimulated by SPA（P﹤0.05）.The mRNA expressions of TLR4,TLR9 and IL-12 in both unblocked group and blocked group were obviously higher than control group（P﹤0.05）,especially Daphnetin of 12.50 μg/ml for strengthening the proliferation of B lymphocytes.The mRNA expressions of TLR4,TLR9 and IL-12 in the blocked group were notably lower than the unblocked group（P﹤0.05）.Conclusion The expression of IL-12 in B lymphocytes can be up-regulated by Daphnetin through natural immunity approach.Daphnetin can enhance immunological functions of B lymphocytes.