重组CFP10-ESAT6蛋白通过TLR信号介导A549细胞抗BCG感染

Recombinant CFP10-ESAT6 protein mediates A549 cells against BCG infection by TLR signal

本研究旨在探究重组CFP10-ESAT6蛋白在结核分支杆菌(Mycobacterium tuberculosis,Mtb)感染肺泡上皮细胞过程中对肺泡上皮细胞TLR信号途径及其介导的炎症反应的影响。利用原核表达系统表达纯化Mtb重组蛋白CFP10-ESAT6,使用重组蛋白处理A549细胞,利用CCK-8试剂盒检测重组CFP10-ESAT6对细胞活性的影响;使用重组CFP10-ESAT6和减毒牛分支杆菌BCG处理细胞并设置相应组别,提取细胞总RNA和总蛋白,利用qRT-PCR技术、Western-blot及ELISA方法分别从转录和翻译水平检测A549细胞中TLR信号途径关键分子及其下游炎症因子的表达变化。结果显示,成功获得高纯度的重组CFP10-ESAT6,并且重组CFP10-ESAT6在一定浓度范围和处理时间条件下能够降低A549细胞的存活率。当BCG或重组CFP10-ESAT6单独处理A549细胞时,细胞中TLR2、TLR4、My D88、TRAF6和NF-κB p65及下游炎症因子IL-6和TNF-α的表达水平显著(P<0.05)或极显著(P<0.01)上调,重组CFP10-ESAT6与BCG共处理时,重组CFP10-ESAT6显著(P<0.05)降低了由BCG刺激所导致的TLR途径关键分子TLR2、TLR4、My D88、TRAF6、NF-κB p65、IL-6及TNF-α等炎症因子的表达水平。上述结果表明,重组CFP10-ESAT6可以减轻A549细胞在抗Mtb感染过程中由TLR信号介导的炎症因子的分泌,这将为进一步加深理解结核病发病机制提供理论依据。

The aim of this study was to investigate the effect of recombinant CFP10-ESAT6 protein on the TLR signaling pathway and its mediated inflammatory response in alveolar epithelial cells during Mycobacterium tuberculosis(Mtb) infection.Mtb recombinant protein CFP10-ESAT6 was purified using a prokaryotic expression system.A549 cells were treated with the recombinant protein,and the effect of recombinant CFP10-ESAT6 on cell activity was detected using CCK-8 kit.The cells were treated with recombinant CFP10-ESAT6 and attenuated bovine M.tuberculosis BCG and set up with corresponding groups.Total RNA and total protein were extracted from the cells.The results showed that high purity recombinant CFP10-ESAT6 was successfully obtained,and recombinant CFP10-ESAT6 was able to reduce the survival rate of A549 cells under certain concentration range and treatment time conditions.When A549 cells were treated with BCG or recombinant CFP10-ESAT6 alone,the expression levels of TLR2,TLR4,MyD 88,TRAF6 and NF-κB p65 and the downstream inflammatory factors IL-6 and TNF-α were significantly(P<0.05) or highly significantly(P<0.01) upregulated in the cells.When recombinant CFP10-ESAT6 was co-treated with BCG,recombinant CFP10-ESAT6 significantly(P<0.05) reduced the expression levels of inflammatory factors such as TLR2,TLR4,MyD 88,TRAF6,NF-κB p65,IL-6 and TNF-α,which are key molecules of TLR pathway caused by BCG stimulation.The above results suggested that recombinant CFP10-ESAT6 can attenuate the secretion of inflammatory factors mediated by TLR signaling in A549 cells during Mtb infection,which will provide a theoretical basis for further deepening the understanding of the pathogenesis of tuberculosis.