GPR160调控结核杆菌入侵宿主巨噬细胞的分子机制研究

It's very important for the research of tuberculosis (TB) pathogenesis and development of new therapy and prevention ways against TB to identify and analyse the key immune regulator factors during the invasion process of Mycobacterium tuberculosis (MTB)to host immune cell. G protein coupled receptors(GPCR)are involved in all kinds of physiological and pathological activities of organism, but the function of GPCRs in the MTB invasion to host are still unknown.It was found from our prior research that: (1) The expression level in peripheral blood mononuclear cell (PBMC) of an orphan receptor-GPR160, which belonging to type A GPCR was much lower than normal subjects; (2) The expression level of GPR160 in macrophage was also down regulated during the early stage of MTB infection; (3)GPR160 mutated macrophage were more susceptible to BCG than normal; (4) After infected with BCG, the CFUs of GPR160 mutated mice were much higher than control mice and lots of acid-free bacillius aggregated distributed. All of the results indicate that GPR160 may involve in the invasion process of MTB to host macrophage and play the protection function. In this project,the function and mechanism of how MTB regulates the expression of GPR160 and GPR160 regulates the MTB invation to macrophage will be analysed from molecular, cellular, animal infection and SNP detection level, etc., furthermore, the influence of GPR160 to the virulence of MTB will be detected, which can provide new ideas for the TB pathogenesis research.

鉴定和分析结核菌入侵宿主免疫细胞过程中关键的免疫调控因子对研究结核病发病机制、开发新的治疗和预防手段十分重要。G蛋白偶联受体（GPCR）参与调控机体各种生理和病理活动，但在结核菌入侵机体中作用尚未见报道。本项目前期研究发现：（1）属于A类GPCR的一个孤儿受体GPR160，在结核病人外周血单核细胞的表达水平明显低于正常人；（2）结核菌H37Rv感染早期明显下调巨噬细胞GPR160的表达；（3）GPR160突变巨噬细胞更容易被BCG入侵；（4）GPR160突变鼠经BCG感染后，荷菌量显著高于对照鼠，抗酸杆菌大量聚集分布,均提示GPR160可能参与结核菌入侵宿主巨噬细胞的过程，并发挥保护作用。本项目将从分子、细胞、动物感染、SNP检测等水平，分析结核菌调控GPR160表达及GPR160调控结核菌入侵巨噬细胞的作用和机制，进而检测GPR160对结核菌致病力的影响，为结核病发病机制研究提供新思路。

结核分枝杆菌可入侵宿主巨噬细胞，调控机体免疫应答，从而逃避宿主免疫监视和药物攻击。G蛋白耦联受体（GPCRs）广泛地参与细胞的各类生理和病理活动的调控，但其在结核分枝杆菌入侵宿主细胞中的功能尚不明确。本室前期工作发现在BCG感染的巨噬细胞中，一种GPCR家族的孤儿受体――GPR160的mRNA和蛋白表达水平早期显著上调。在此基础上，本项目从4个方面开展了研究：分枝杆菌如何调控GPR160的表达，GPR160调控分枝杆菌入侵宿主巨噬细胞的作用和机制，GPR160对分枝杆菌感染致病力的影响，GPR160对巨噬细胞吞噬非分枝杆菌的作用。结果发现，分枝杆菌通过其主要毒力因子LPS,PGN早期上调GPR160表达。从GPR160突变鼠分离的腹腔巨噬细胞进行BCG感染实验，与对照细胞相比，BCG入侵水平显著下调，通过特异性siRNA沉默以及小鼠感染实验，均获得相同结果。与对照细胞相比，BCG诱导GPR160突变巨噬细胞ERK信号分子的磷酸化水平显著下调。通过特异性抑制剂或siRNA抑制ERK的表达，也可显著下调BCG的入侵。动物感染实验发现GPR160突变小鼠被BCG感染后，荷菌量低于对照鼠，病理切片也明显减轻。进一步研究还发现，GPR160突变巨噬细胞对大肠杆菌和土拉杆菌的吞噬水平也显著下调。上述研究结果均提示，GPR160可通过上调ERK信号通路在BCG入侵宿主巨噬细胞的过程中发挥重要的调控作用。研究结果为探讨结核病的发病机制以及GPCR在结核感染免疫甚至慢性感染性疾病中的作用提供新的思路和基础，为以GPCRs为药物靶点，开发新的抗结核药物提供了理论依据。