TLR2介导鸡毒支原体脂质相关膜蛋白诱导DF-1细胞表达IL-1β的研究

Lipid-associated membrane proteins from Mycoplasma gallisepticum inducing DF-1 cells to express IL-1β though TLR2

Toll样受体2(TLR2)在鸡毒支原体(MG)诱导天然免疫反应机制中的作用尚未明确。为鉴定TLR2在MG脂质相关膜蛋白(LAMPs)诱导细胞表达IL-1β中的作用,本研究通过RT-PCR和ELISA方法检测LAMPs刺激后DF-1细胞中IL-1β的m RNA和蛋白水平,结果显示LAMPs可以诱导DF-1细胞分泌IL-1β,并确定最佳刺激时间为6 h,最佳刺激剂量为1μg/m L。此外,通过构建TLR2重组表达质粒p CMV-HA-TLR2,并将该质粒转染DF-1细胞进行过表达及通过抗体封闭TLR2的两种处理方式,处理后采用LAMPs刺激细胞。经检测显示:TLR2过表达处理组的IL-1βm RNA和蛋白水平显著升高,而抗体处理组的IL-1βm RNA和蛋白水平下调。结果表明,LAMPs能够与DF-1细胞膜上的TLR2受体结合,有效的诱导DF-1细胞表达并分泌IL-1β,而且IL-1β的释放可能与天然免疫系统的经典通路即NF-κB信号通路调控有关。本研究为阐述MG的致病机制提供相关实验依据。

Toll like receptors 2 （TLR2） is a pattern recognition receptors, which recognize conserved microbial structures, such as bacterial lipopolysaccharides, and activate signaling pathways that result in immune responses against microbial infections. In present, the role of TLR2 against Mycoplasma gallisepticum infection mechanism is still unknow. To study the involvement of TLR2 in lipid-associated membrane proteins （LAMPs） of M.gallisepticum induced IL-1β expressing, the expression level of IL-1β was detected by RT-PCR and ELISA in the DF-1 cells stimulated with the LAMPs. Initially, the LAMPs were proved to be able to efficiently induce the expression of IL-lβ, and the optimized concentration and time of LAMPs to stimulate the expression of IL-1β mRNA were lμg/mL for 6 hrs. In addition, DF-1 cells were transfected with recombinant plasmid of pCMV-HA-TLR2 for over expression of the TLR2 or the cells pretreated with anti-TLR2 monclonal antibody （MAb）, respectively, and then treated with LAMPs. The results showed that the expression of IL-lβ mRNA and IL-lβ were found to be upregulated in the TLR2 overexpression group, but it was deregulated the expression of IL-1 β mRNA and IL-1βin the anti-TLR2 MAb pretreated group. Those data demonstrate that LAMPs have the ability to efficiently induce the IL-lβ expression in DF-1 cells and TLR2 plays an important role in this process.