结核分枝杆菌分泌蛋白TB8.4和MTP64通过TLR2调控巨噬细胞功能的研究

China proves to be one area with high burden of tuberculosis， and various kinds of secreted protein from Mycobacterium tuberculosis have drawn an increasing amount of attention from scholars globally during the process of the diagnosis and vaccine design for this disease. During the pathogen caused infection within the host, influence to the macrophages caused by the secreted protein considerably worth focus. In this project, we find two secreted proteins TB8.4 and MPT64 may have the function to activate macrophages,so the cytokines secretion and the expression of MHC class II as well as CD80 and CD86 in the surface of macrophage induced by them will be detected first. Additionally, the antigen processing and presentation assay will be carried out to identify the ability of macrophages to present antigens ,and the effects that protein vaccines have on the immune responses of T cells.Then neutralizing Abs against TLR2 and TLR2-/- mouse will be used to investigate the relationship between TLR2 and the changes of macrophages induced by the secreted proteins. At last, Western blotting and the specific inhibitors will be provided to observe the activation of NF-kB, ERK1/2, p38, and JNK in macrophages,the molecular mechanisms will be futher explored as well.It is highly helpful to gain a deeper understanding of the relationship between the innate immunity and the adaptive immunity by exploring the regulation effect to the macrophages exerted from the secreted protein.Besides, insights for vaccine development and clinical diagnosis can be procured through this investigation as well.

中国是结核高发地区之一，结核分枝杆菌分泌蛋白在结核的诊断和疫苗研发过程中一直是研究的热点。在这些候选疫苗蛋白的筛选过程中，研究它们如何影响宿主巨噬细胞的功能至关重要。在本课题中，我们发现两个重要的分泌蛋白TB8.4和MPT64可能通过TLR2刺激巨噬细胞活化，拟研究它们对巨噬细胞细胞因子释放、表面MHCII类分子和共刺激分子CD80、CD86表达情况的影响，然后通过抗原呈递实验测定巨噬细胞抗原提呈能力的变化以及对T细胞功能的影响，利用抗体阻断及基因敲除小鼠来检测TLR2与巨噬细胞产生这些变化的相关性。最后，通过阻断剂处理后Western blot检测TLR2下游信号分子NF-kB及ERK1/2、p38、JNK的活化程度，并且对进一步的分子机制进行探讨。通过探讨分泌蛋白对巨噬细胞功能的调节有助于理解结核分枝杆菌感染过程中固有免疫与适应性免疫之间的联系，为后续的疫苗开发和临床诊断提供依据。

中国是结核高发地区之一，结核分枝杆菌分泌蛋白在结核的诊断和疫苗研发过程中一直是研究的热点。在这些候选疫苗蛋白的筛选过程中，研究它们如何影响宿主巨噬细胞的功能至关重要。本研究主要对结核分枝杆菌分泌蛋白MPT64和TB8.4对巨噬细胞和树突状细胞的功能影响以及作用机制进行了研究。本课题完成的工作内容包括：1：MPT64和TB8.4均能刺激RAW264.7细胞和树突状细胞分泌IL-6和TNF-α，且上调巨噬细胞表面CD80，CD86，MHC-I和MHC-II分子的表达，以及TLR2和TLR4的表达。2：通过阻断抗体以及TLR2/TLR4基因敲除小鼠测定分泌蛋白MPT64和TB8.4对巨噬细胞功能的影响发现，MPT64对巨噬细胞功能的影响依赖TLR2和TLR4，TB8.4主要依赖TLR2调控巨噬细胞的功能。3：通过western blot实验，选择MAPK信号通路关键的节点分子探讨了MPT64和TB8.4引起巨噬细胞功能变化所涉及的信号通路，发现MPT64所参与的信号通路与P38 MAPK、JNK具有联系，TB8.4所参与的信号通路与P38 MAPK、JNK、ERK均有关系。两种目的蛋白都可以促进巨噬细胞的IκBα的降解，即NF-κB活化，从而促进细胞因子的分泌。4：MPT64和TB8.4能够增强巨噬细胞和DC的抗原提呈能力，进而促进CD8+T细胞的增殖和活化。5：荷瘤小鼠体内实验发现TB8.4能够抑制肿瘤的生长，且抑制作用是通过增加肿瘤内CD8+T细胞、DC细胞和巨噬细胞的比例和功能来起作用。综上所述：通过探讨MPT64和TB8.4对巨噬细胞功能的调节有助于理解结核分枝杆菌感染过程中固有免疫与适应性免疫之间的联系，为后续的疫苗开发和临床诊断提供依据。本研究成果共发表标注基金资助号的SCI论文1篇，中文核心期刊论文1篇；获授权发明专利2项，申请国家发明专利6项；培养获得学位的博士研究生1名，硕士研究生11名。项目投入经费64.96万元，剩余经费15.689073万元，计划用于MPT64和TB8.4蛋白的后续功能研究以及文章发表费用。