不同宿主来源巨噬细胞对问号钩端螺旋体杀菌分子机制研究

Leptospirosis is an important global zoonotic disease. After infection with leptospires, animals such as rats usually have no clinical symptoms and shed spirochetes from their urine for a long period of time, whereas all human individuals fall ill. Our previous study found that mouse macrophages could kill intracellular leptospires, while leptospires in human macrophages were survived and replicated, which maybe associated with the diversity about the leptospiral pathogenicity in human and animals. However, the mechanism about the pathogenetic diversity remains unknown. Recently, the activation of reactive oxygen intermediates (ROIS), reactive nitrogen intermediates (RNIS), natural resistance associated macrophage protein l (Nramp l) and acidification of phagocytotic vesicles have been shown to determine whether macrophages kill many intracellular bacteria. In this research, we take the major bactericidal pathways and their acting mechanisms as the entry points to determine the effectiveness and diversity in activated levels of ROIS, RNIS and Nramp l systems, and acidifying level of phagocytotic vesicles during killing of intracellular leptospires in human and mouse macrophages by using laser confocal microscopy, electron microscopy, flow cytometry and multiple molecular biological techniques. Subsequently, the correlation as well as molecular mechanism of the functional status of bactericidal routes with the killing, survival or replication of leptospires in macrophages from different host origins will be determined. The results of this study will contribute to elucidate pathogenic mechanism of Leptosira as well as provide novel ideas for prevention and therapy of leptospirosis, which referring to original findings with high academic value.

钩端螺旋体（简称钩体）病是全球流行的重要人兽共患病。钩体侵入鼠类等动物后常无症状且长期尿液排菌，人感染钩体后均发病。我们发现的小鼠巨噬细胞能杀灭钩体、人巨噬细胞内钩体在胞浆中存活并繁殖的现象可能与钩体对人和动物致病差异性有关，但机制不明。近年发现活性氧和活性氮中间产物（ROIS、RNIS）、巨噬细胞蛋白l（Nramp l）活化及吞噬泡酸化与巨噬细胞能否杀灭胞内菌密切相关。本项目拟以不同宿主巨噬细胞吞噬钩体后主要杀菌途径及作用机制为切入点，采用激光共聚焦、流式细胞术、电镜及多种分子生物学技术，了解人和小鼠巨噬细胞吞噬钩体后ROIS、RNIS、Nramp l系统活化和吞噬泡酸化程度及其在杀灭钩体中的作用与差异，确定上述杀菌途径功能状态与钩体在不同宿主来源巨噬细胞内被杀灭或生存繁殖的关系及分子机制，研究结果不仅有助于深入阐明钩体致病机制，也为钩体病防治提供新思路，具有较高创新性和学术价值。

钩端螺旋体（简称钩体）病是全球流行的重要人兽共患病。钩体侵入鼠类等动物后常无症状且长期尿液排菌形成传染源，人感染钩体后均发病，但钩体感染致人和宿主动物呈现不同结局的现象迄今没有科学解释。我们以往研究发现的小鼠巨噬细胞能杀灭钩体、人巨噬细胞内钩体在胞浆中存活并繁殖的现象可能与钩体对人和动物致病差异性有关，但机制不明。在国家自然科学基金的资助下我们研究了人和小鼠巨噬细胞噬钩体后ROIS、RNIS、Nramp l系统活化和吞噬泡酸化程度，研究了上述杀菌途径功能状态与钩体在不同宿主来源巨噬细胞内被杀灭或生存繁殖的关系及分子机制，发现问号钩体感人和小鼠巨噬细胞后ROS和超氧离子水、iNOS mRNA和蛋白表达水平、NO均显著上调，但以小鼠巨噬细胞上调更明显。ROIS和RNIS杀菌途径被阻断后，人和小鼠巨噬细胞内钩体与溶酶体融合减少，吞噬泡内钩体数目减少，细胞质内钩体数量及钩体活性明显增加，但小鼠巨噬细胞内钩体的以上变化更为明显。此外，研究发现Nramp-1和胞内吞噬泡酸化途径对钩体胞内命运无明显影响。究结果提示，ROIS和RNIS途径均参与了人和小鼠巨噬细胞对胞内钩体的杀菌作用，且以小鼠巨噬细胞内的杀菌作用更为明显，对解释钩体感染致人和宿主动物呈现不同结局的现象提供了科学依据。本研究共发表核心期刊论文6篇，另有2篇SCI论文正在审稿过程中。本人受邀请在第九届国际钩体病学会学术会议上做了大会报告，培养硕士研究生1名。对照项目计划书，我已完成了项目的研究任务并达到了考核要求。