自噬作为一种新发现的抗胞内病原体机制，可有效清除胞内结核杆菌。目前结核杆菌调节细胞自噬的研究甚少，其分子机制尚未明确。我们前期筛选实验发现四种结核杆菌分泌型毒力因子可抑制自噬流量，具体表现为抑制自噬体形成和阻断自噬体降解，因此，本研究提出结核杆菌可通过分泌型毒力因子调节自噬的假说。为明确该调节的具体分子机制，本课题组已构建了稳定表达GFP-LC3及mRFP-GFP-LC3报告基因的巨噬细胞系，将应用蛋白激酶抑制剂特异性阻断已明确的自噬调控信号通路，并结合免疫印迹、实时定量PCR及电镜技术，明确这四种毒力因子所参与调控自噬的信号通路；应用RNA干扰、免疫共沉淀、激光共聚焦及自噬体-溶酶体融合阻断实验，研究毒力因子在自噬体形成及自噬体-溶酶体融合环节所作用的靶点。本研究对阐明结核杆菌调节宿主细胞自噬的分子机制具有重要意义，将为免疫干预或靶向药物开发提供新思路。

As a new defence mechanism for control of intracellular pathogens, autophagy can promote the elimination of mycobacterium tuberculosis. So far, the underlying mechanism used by the intracellular bacteria to regulate autophagy is little known. Our preliminary experiments showed that four secreted virulence factors inhibited autophagy flux by inhibiting autophagosome formation and blocking autophagosome degradation. This indicated a hypothesis that autophagy could be regulated by secreted virulence factors from M. tuberculosis. To dissect the regulation mechanism used by the virulence factors, we have established stable lines of macrophage expressing GFP-LC3 or mRFP-GFP-LC3. These cells will be subjected to specific protein kinase inhibitors to block autophagic pathway, using western-blotting, real-time quantitative PCR and transmission electron microscopy assays to define which pathway is regulated by these virulence factors, respectively. To define the targets of these virulence factors in the processes of autophagosomal formation and autophagosomal-lysosomal fusion, these cells will be subjected to RNAi, immunoprecipitation, confocal microscopy and autophagosomal-lysosomal fusion block assays. This study will provide important insight into the mechanisms underlying autophagy regulation of host cells by M. tuberculosis and contribute to the development of targeted drugs or immunologic intervention.