【作者】 刘淑清；

【导师】 朱鸿飞；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2014， 博士

【摘要】 牛分枝杆菌的主要宿主细胞是巨噬细胞，该菌入侵机体后通过TLR传递分子信号激活丝裂原蛋白激酶（MAPK），最终激活核转录因子（NF-κB），激活巨噬细胞免疫应答的关键分子，上调IL-1、IL-6和TNF-等炎症因子的转录和表达，激活机体的先天免疫应答。为探讨TB10.4是否通过TLR调控炎症反应及相关信号通路的信号转导，本研究开展了以下工作：首先通过ELISA检测rTB10.4对RAW264.7细胞TNF-.、IL-6和IL-12p40细胞因子表达的影响，结果显示rTB10.4能够显著激活TNF-、IL-6和IL-12p40的表达，并且呈剂量依赖关系。采用TLR2和TLR4的抗体对RAW264.7细胞进行预处理，结果显示TLR2抗体可以显著抑制rTB10.4诱导RAW264.7细胞表达TNF-、IL-6和IL-12p40，而TLR4抗体无此抑制作用。表明rTB10.4诱导RAW264.7细胞产生TNF、IL-6和IL-12p40可能是依赖TLR2通路传递信号。采用Western Blot和量化流式细胞分析仪检测rTB10.4诱导细胞因子的表达是否与MAPK信号通路的激活有关，结果显示，rTB10.4能够诱导RAW264.7细胞激活p38和ERK1/2信号通路，并且诱导RAW264.7细胞发生明显的p38及ERK核转位；但rTB10.4不能激活RAW264.7细胞中JNK信号通路磷酸化。通过使用TLR-2及TLR-4抗体预处理RAW264.7细胞，评估rTB10.4激活RAW264.7细胞中p38和ERK1/2磷酸化是否通过TLR2和TLR4信号通路，结果显示TLR-2抗体显著降低rTB10.4诱导的p38和ERK1/2磷酸化；p38激酶的特异性抑制剂SB203580和MAPK激酶1/2特异性抑制剂U0126能够显著抑制rTB10.4诱导RAW264.7细胞分泌TNF-、IL-6和IL-12p40细胞因子。进一步采用NF-κB荧光素酶报告系统、Western Blot和量化流式分析仪检测rTB10.4诱导细胞因子的表达是否依赖NF-κB信号通路，结果表明rTB10.4在刺激RAW264.7细胞后12h和24h能显著激活NF-κB，并通过诱导IκB的降解激活p65的磷酸化，诱导RAW264.7细胞中NF-κBp65发生明显核转位。NF-κB特异性抑制剂BAY11-7082作用结果表明：rTB10.4蛋白刺激RAW264.7细胞分泌表达TNF-、IL-12p40和IL-6依赖NF-κB信号通路。综上所述，本研究首次研究发现了rTB10.4能够上调RAW264.7细胞TNF-、IL-6和IL-12p40的表达，并且这一过程是通过TLR2受体介导p38MAPK、ERK1/2和NF-κB信号通路实现。该研究为牛结核病感染与诊断提供了分子基础，并且对理解牛分枝杆菌与宿主细胞的相互作用及分子机制提供新的研究思路。更多还原

【Abstract】 Macrophages play a significant role in the host defense mechanism of Mycobacterium bovis,Toll-like receptors (TLR) are key molecules involved in activating macrophages, Several proteinkinases, such as mitogen-activated protein kinases (MAPKs) and the nuclear translocation of theNuclear Factor-Kappa B (NF-κB) are also activated via the TLR signaling pathway，stimulating thetranscription and production of IL-1, IL-6and TNF-, activating the innate immune response. Toevaluate whether rTB10.4interact with Toll-like receptors (TLRs) and the signaling pathways for activeinflammation, this study has carried out mainly works as the following aspects:Firstly, we tested the activation role of M. bovis-derived recombinant TB10.4protein (rTB10.4) onthe expression of TNF-, IL-6, and IL-12p40in RAW264.7cells by ELISA. The result showed thatRAW264.7cells, exposed to rTB10.4, produced significantly higher levels of TNF-, IL-6, and IL-12p40in a dose-dependent manner than control groups. Furthermore, the expressions of TNF-, IL-6, andIL-12p40were inhibited in RAW264.7cells when TLR2signals were blockaded by treating withanti-TLR2. However, TLR4had no such effects after treating with anti-TLR4. These data suggested thatthe activation role of rTB10.4on expression of cytokines was mediated by TLR2signals.We examined whether rTB10.4could activate the MAPK pathway and then effect the expressionsof cytokines by the methods of Western Blot and Image Stream100. Our results showed that rTB10.4could activate the p38kinase (p38) and extracellular-regulated kinase (ERK) signals and induce thenuclear translocation of p38and ERK. However, rTB10.4had no effects on phosphorylation of JNKpathway. After treated with anti-TLR-2, the phosphorylations of p38and ERK1/2were significantlyreduced in RAW264.7cells. The inhibition of p38and ERK activity with inhibitor of SB203580(p38)or U0126(ERK1/2) had a significant effect on rTB10.4-induced secretion of TNF-, IL-6, and IL-12p40.To investigate whether TLR2could contribute to rTB10.4induces the activation of NF-κB byNF-κB luciferase activity detection. RAW264.7cells transfected with a luciferase reporter generevealed that rTB10.4stimulation significantly activated the NF-κB luciferase activity. Then wemeasured the rTB10.4-induced phosphorylation and nuclear translocation of NF-κB p65by Westernblot analysis，these data suggest that rTB10.4mediates the activation of NF-κB p65by triggering thedegradation of IκB in the cytoplasm in time-dependent manner. Then, we detected the ranscriptionalcompetence of nuclear NF-κB p65by rTB10.4stimulation using the ImageStream analysis, the resultsshowed that translocation of NF-κB p65was observely induced by rTB10.4. Furthmore, we pre-treatedRAW264.7cells with a specific NF-κB inhibitor BAY-117082. The production of TNF-or IL-12p40dramatically blocked by BAY-117082. These observations suggested that TB10.4induced theproduction of TNF-, IL-12p40, and IL-6via the NF-κBsignalling pathway.In summary, our findings provide evidence that rTB10.4promotes the production of TNF-, IL-6,and IL-12p40in RAW264.7cells via TLR2, and that the p38MAPK, ERK1/2and NF-κB pathways were essential to this process. Thus, our studies provide a better understanding of the molecularmechanisms influencing host/pathogen interactions necessary to prevent Mycobacterium bovisinfection.更多还原