结核分枝杆菌CFP10和ESAT6蛋白在巨噬细胞抗结核分枝杆菌感染中的调控作用及分子机制研究

Tuberculosis(TB), caused by the infection of Mycobacterium tuberculosis (Mtb), is one of the world’s leading infectious diseases and major public health problem in the world. The host-bacterial interaction plays important roles in the development of TB. Alveolar macrophages are the main target cells of Mtb, which are able to provide critical intracellular niches for Mtb establishing infection in host. The mode of cell death of macrophages following Mtb infection is thought to play a decisive role in the pathogenesis of TB, in which the apoptotic death of the infected cells is likely associated with the elimination of Mtb, while necrotic cell death may facilitate the dissemination and transmission of the pathogens. Therefore, the balance of these two modes of cell deaths in macrophages may be an important mechanism that controls the course of Mtb infection. It has been demonstrated that CFP10 and ESAT6, which are immune-protective antigens encoded by region of difference-1 that are absent in BCG but are present in virulent strains of Mtb and Mycobacterium bovis, may function as a regulator of macrophage cell death during Mtb infection. In this study, murine alveolar macrophage RAW264.7 was used to investigate the regulation and molecular mechanisms of CFP10 and ESAT6 on macrophages in response to Mtb infection. The phagocytosis of macrophage, inflammatory cytokines and apoptotic characteristics of macrophage were ascertained by qRT-PCR, Western blot, ELISA, flow cytometry method, electron microscope and laser confocal technology. Hopefully, this study will lay a foundation for fully understand the mechanisms of anti-TB immunity, especially mechanisms of immune evasion by Mtb and molecular pathogenesis of Mtb, and we also hope the results of this study can provide new targets for the development of new drugs and vaccines for tuberculosis treatment.

结核病是由结核分枝杆菌感染引起的慢性致死性人兽共患性传染病，该病的流行给人类公共卫生安全造成严重威胁。宿主与病原相互作用所致的炎性反应、巨噬细胞凋亡与坏死对结核病的发生发展具有重要作用。肺泡巨噬细胞作为结核分枝杆菌感染的靶细胞在抗结核免疫中发挥重要作用。研究证实，结核分枝杆菌蛋白CFP10和ESAT6对巨噬细胞具有调控作用。因此，本研究拟以小鼠肺泡巨噬细胞系RAW264.7为研究对象，通过实时荧光定量PCR、蛋白免疫印迹、流式细胞术和激光共聚焦技术等手段研究结核分枝杆菌CFP10、ESAT6蛋白及CFP10-ESAT6融合蛋白刺激巨噬细胞后对巨噬吞噬能力、巨噬细胞炎性反应以及巨噬细胞凋亡等的调控作用，分析其调控机理，探讨它们在结核分枝杆菌致病过程中的地位和所起的作用，从而加深对结核分枝杆菌致病机理及机体抗结核免疫机制的了解，为研制结核病治疗药物和新型疫苗提供新的靶点。

由结核分枝杆菌（Mtb）感染引起的结核病是目前危害人类健康的一个重要的公共卫生问题。肺泡巨噬细胞和肺泡上皮细胞作为Mtb感染的宿主细胞和攻击的靶细胞，其命运对结核病的发生、发展具有非常重要的影响。CFP10和ESAT6是Mtb的免疫保护性抗原，它们在卡介苗BCG中的缺失被认为是BCG减毒的主要原因。本项目研究了Mtb蛋白CFP10和ESAT6对肺泡巨噬细胞和肺泡上皮细胞炎性反应和凋亡的影响及调控机制，取得了以下重要研究成果：1）构建了3个表达CFP10和ESAT6蛋白的真核表达载体和3个表达CFP10和ESAT6蛋白的重组腺病毒载体，这些载体的构建为在细胞模型中以及后续在动物模型中深入研究CFP10和ESAT6在Mtb致病中的作用及其分子机制奠定了良好的基础；2）构建了表达CFP10和ESAT6的原核表达载体，制备并纯化获得了纯度较高的CFP10和ESAT6蛋白，研究了它们对肺泡巨噬细胞和肺泡上皮细胞炎性反应及细胞凋亡的影响；3）阐明了CFP10和ESAT6对肺泡巨噬细胞和肺泡上皮细胞炎性反应及凋亡的调控机制。以上结果表明，CFP10和ESAT6在Mtb感染肺泡巨噬细胞和肺泡上皮细胞过程中对宿主细胞炎性反应和凋亡具有重要的调控作用，而TLRs/MyD88-NF-κB信号通路和NLRP3、NLRC4及AIM2炎性小体介导的炎性反应以及内质网应激介导的细胞凋亡在肺泡巨噬细胞和肺泡上皮细胞应答Mtb的免疫调控中发挥了重要作用。这些发现加深了我们对Mtb致病机理及机体抗Mtb免疫机制的了解，为全面了解Mtb与肺泡巨噬细胞和肺泡上皮细胞的复杂相互作用过程奠定基础。