Shh/NF-κB/p53信号通路在肺泡Ⅱ型上皮细胞抗结核分枝杆菌免疫应答中的调控作用

Along with alveolar macrophages, the alveolar epithelial Type Ⅱ (AECⅡ) cells are the first targets of Mycobacterium tuberculosis (Mtb) infection in the lung. However, the role of AECⅡ cells in the immunological regulation against Mtb remains largely unknown. And other studies show that the Sonic hedgehog (Shh) and p53 signaling pathways play an important role in the regulation of immunity of host cells in response to pathogens via NF-κB signaling pathway. Therefore, the proposal is based on our recent analysis of the de novo transcriptome sequencing of Mtb infected animal model lungs, a novel AECⅡculture model and Wnt signaling in immune regulation, and aim to explore the immunoregulatory role of Shh signaling in AECⅡ cells of against avirulent BCG and virulent H37Rv Mtb infection by asscessing the Shh signaling molecules and tuberculosis related pro-inflammatory factors which are determined by a quantitative real-time PCR assay, in an interaction model of human AECⅡ cells and mycobacteria (BCG or H37Rv); meanwhile, the croostalks of the Shh/NF-κB, p53/NF-κB and Shh/NF-κB/p53 signaling pathways upon the infection of Mtb will also be demonstrated by assays of transcriptome microarray, PCR-Array and immunoblot and other techniques, and these results will be further verified in the Shh and p53 gene knock-out mouse. An accomplishment of these studies will allow us better understand the molecular mechanisms of AECⅡ cells in response to Mtb infection , as well as the interaction of host and pathogen between Mtb and AECⅡ cells. These results will lay a foundation for the further understanding the pathogenesis of targeting agents against Mtb infection.

结核分枝杆菌(Mtb)感染肺脏时，肺泡巨噬细胞和肺泡Ⅱ型上皮细胞(AECⅡ)首先受到侵染，但目前AECⅡ抗Mtb免疫调控作用尚不明确。另有研究表明，传统的Shh、p53和NF-κB信号通路间存在多层次cross-talk关系，并以此来参与调控机体的先天免疫应答与炎症反应来对抗病原菌的感染。因此，本项目拟在Mtb感染动物模型肺脏转录组测序、AECⅡ-ROCK抑制剂培养和Wnt信号调控等研究基础上，以Mtb与AECⅡ互作为模型，结合Shh和p53 KO小鼠模型，分析AECⅡ中Shh/NF-κB、p53/NF-κB和Shh/NF-κB/p53信号通路在抗Mtb先天免疫反应中的cross-talk作用模式，揭示Shh、NF-κB与p53信号通路在AECⅡ与机体抗Mtb免疫应答中的调控作用，从而阐明Mtb与AECⅡ的病原-宿主相互作用机理，为深入研究机体抗Mtb感染的免疫调控机制奠定一定的理论基础。

结核分枝杆菌(Mtb)感染肺脏时，肺泡巨噬细胞和肺泡Ⅱ型上皮细胞(AECⅡ)首先受到侵染，但目前AECⅡ抗Mtb免疫调控作用尚不明确。研究发现，在机体通过先天免疫应答与炎症反应来对抗病原菌的感染过程中，Shh、p53和NF-κB信号通路间通过多层次相互作用参了与调控。因此本项目通过建立Mtb与AECⅡ相互作用模型，结合小鼠BCG感染模型，开展了以下的研究内容：①利用Mtb感染AECⅡ细胞A549，分析了Shh和NF-κB信号通路Shh、Ptch、Gli1、p-NF-κB等相关信号分子的变化以及IL8、TNF-α等炎症细胞因子的变化规律；建立BCG与过表达Shh和NF-κB基因的AECⅡ互作模型，分析了Hedgehog（Hh）和NF-κB信号通路信号分子Shh、Gli1、p-NF-κB等及炎症细胞因子IL-6、IL-8和TNF-α等的表达变化规律，解析Hh和NF-κB信号通路“crosstalk”关系。②建立了BCG与p53和NF-κB基因过表达和干扰的AECⅡ互作模型，探讨了p53和NF-κB信号通路中TLR-4、TRAF6、p53、MDM2/p-MDM2和CBP等信号分子和炎症细胞因子IL-6、IL-8、TNF-α和IFN-γ等的表达情况，阐释了在Mtb感染AECII中p53信号通路如何发挥了对NF-κB信号通路的调控作用并调控炎症细胞因子的分泌。③建立了BCG感染Shh干扰和过表达的小鼠肺脏上皮细胞TC-1模型，分析了TC-1上清液对RAW264.7中p53与NF-κB信号通路相关信号及炎症细胞因子分泌的调控，并建立小鼠BCG感染模型，观察小鼠肺脏与脾脏病理变化及Shh、p53和NF-κB信号通路相关因子的变化，在体内外水平揭示了Shh、p53和NF-κB信号通路在调控免疫相关细胞抗BCG感染时的免疫调控作用。④分别对3个健康人和结核病患者的外周血转录组进行了测序分析，发现了33个差异表达基因，其中12个上调，21个下调。通过以上研究内容的完成，从细胞和动物水平揭示了Mtb感染AECⅡ细胞时，Shh信号通路与p53信号通路之间的相互作用关系，并通过调控NF-κB信号通路相关信号分子与炎症细胞因子的分泌等在病原-宿主相互作用中参与先天免疫调控，从而为进一步研究AECⅡ细胞抗结核免疫的分子机制奠定了理论基础。