烟曲霉对人中性粒细胞结构和功能的影响
发布时间：2018-11-16 09:37
【摘要】： 烟曲霉是自然界广泛存在的真菌,可以引起免疫功能低下的病人,尤其是进行了骨髓移植或器官移植的病人严重的机会感染。因为恶性肿瘤、粒细胞减少或功能障碍、糖皮质类固醇和免疫抑制剂的使用等原因造成的患者免疫功能缺陷,是侵袭性曲霉病的高危因素。近年来,侵袭性曲霉病发病率不断上升,并成为免疫抑制患者死亡的主要原因之一。对于免疫功能正常的人来说,它可以被中性粒细胞(PMN)杀灭并清除。PMN是人体防御系统的重要组成部分,与机体的非特异性抗感染过程有关,该类细胞具有强大的吞噬杀菌功能,其作用过程历经趋化运动,识别并结合吞噬物质,进而摄入胞内,最终杀伤并降解病原体。然而烟曲霉孢子与中性粒细胞之间的关系尚未完全清楚。 本实验以PMN为研究靶细胞,主要以烟曲霉孢子及其代谢产物对它的刺激,分别观察PMN的形态学、中性粒细胞呼吸爆发产生O2-水平以及吞噬率和吞噬指数的改变,为进一步探讨器官移植病人易患烟曲霉病的发病机制的研究奠定基础。 我们首先用改良法分离和纯化正常人和肝移植患者的PMN,姬姆萨染液示PMN的纯度较高,其比例98%,台盼蓝排斥法测活细胞98%。此法能够既简单又快速获得中性粒细胞且对其活性影响较小。在此基础上,将中性粒细胞与烟曲霉孢子及其代谢产物共孵育,利用PAS染色和电镜技术观察PMN的形态学改变与吞噬功能的关系;通过扫描电镜和透射电镜结果进一步阐明PMN内部及外部结构的形态学变化。我们发现,正常PMN表面有突起及伪足形成,粒细胞胞浆、胞质密度均匀,线粒体结构完整。在被烟曲霉孢子激活后,PMN的表面积可以明显增大,即可以使一个PMN同时粘附和吞噬多个孢子;同时,在细胞内吞过程中,细胞内溶酶体和内质网等细胞器可明显增多。烟曲霉的代谢产物----烟曲霉渗出物(AfD)对PMN存在一定细胞毒性作用,这在促进烟曲霉感染的发展中可能起着重要的作用。 O2-呼吸爆发是PMN激活后行使杀菌功能的主要途径之一。运用流式细胞仪检测中性粒细胞呼吸爆发产生O2-水平可以间接地反映PMN的吞噬功能,结果显示肝移植患者PMN经孢子混合液刺激后,其O2-的呼吸爆发水平明显低于经孢子悬液刺激的结果。通过烟曲霉孢子及AfD刺激正常人和肝移植患者PMN,利用PAS染色和电镜技术对其吞噬功能的变化进行研究发现肝移植患者PMN吞噬烟曲霉孢子的吞噬率和吞噬指数低于正常人;患者PMN经孢子混合液刺激后,其吞噬率和吞噬指数低于经孢子混悬液刺激的结果。 通过以上研究,肝移植患者易感烟曲霉菌与其PMN的呼吸爆发水平较低有关,也进一步证明了正常人和肝移植患者PMN的形态学结构和其功能的相关性。
[Abstract]:Aspergillus fumigatus (Aspergillus fumigatus) is a widespread fungus in nature, which can cause severe opportunistic infection in patients with low immune function, especially in patients undergoing bone marrow transplantation or organ transplantation. Because of malignant tumor, granulocytopenia or dysfunction, and the use of glucocorticoid and immunosuppressant, the patients with immune deficiency are the high risk factors of invasive aspergillosis. In recent years, the incidence of invasive aspergillosis has been increasing and has become one of the main causes of death in immunosuppressive patients. For people with normal immune function, it can be killed and cleared by neutrophil (PMN). PMN is an important part of the human defense system and is related to the body's nonspecific anti-infection process. This kind of cells have powerful phagocytic and bactericidal function, its action process goes through chemotaxis movement, recognize and combine phagocytic substance, then ingest the intracellular, kill and degrade pathogens. However, the relationship between Aspergillus fumigatus spores and neutrophils has not been fully understood. In this study, PMN was used as the target cell, and the PMN was stimulated by Aspergillus fumigatus spores and its metabolites. The morphology of PMN, the changes of O _ 2-level, phagocytosis rate and phagocytic index of neutrophil respiratory burst were observed, respectively. To further study the pathogenesis of aspergillosis in organ transplant patients. We first used the improved method to isolate and purify the PMN, Giemsa dye from normal and liver transplantation patients. The purity of PMN was high. The proportion of PMN was 98. The living cells were measured by trypan blue rejection method. This method is simple and rapid to obtain neutrophils and has little effect on its activity. On this basis, neutrophils were incubated with Aspergillus fumigatus spores and their metabolites, and the relationship between morphological changes and phagocytosis of PMN was observed by PAS staining and electron microscopy. The morphological changes of internal and external structures of PMN were further elucidated by SEM and TEM results. We found that there were processes and pseudopods on the surface of normal PMN, granulocyte cytoplasm, homogeneous cytoplasmic density and intact mitochondrial structure. After activated by Aspergillus fumigatus spore, the surface area of PMN could increase significantly, that is, one PMN could adhere to and swallow many spores at the same time, meanwhile, during the process of endocytosis, the cellular organelles such as lysosome and endoplasmic reticulum could increase obviously. (AfD), the metabolite of Aspergillus fumigatus, has a cytotoxic effect on PMN, which may play an important role in promoting the development of Aspergillus fumigatus infection. O-2-respiratory burst is one of the main ways to perform bactericidal function after PMN activation. Flow cytometry was used to detect the level of O _ 2-produced by neutrophil respiratory burst. The results showed that the phagocytic function of PMN could be indirectly reflected by the level of O _ 2 -. The results showed that PMN was stimulated by mixed spore solution in liver transplantation patients. The respiratory burst level of O-2-was significantly lower than that stimulated by spore suspension. The changes of phagocytic function of Aspergillus fumigatus spores and AfD stimulated normal persons and liver transplantation patients were studied by PAS staining and electron microscopy. It was found that the phagocytic rate and phagocytic index of PMN were lower than those of normal subjects. The phagocytosis rate and phagocytic index of PMN stimulated by spore mixture were lower than that of spore suspension. According to the above studies, the susceptibility to Aspergillus fumigatus in liver transplantation patients is related to the lower level of respiratory burst of PMN in patients with liver transplantation, which further proves the correlation between morphological structure and function of PMN in normal persons and liver transplantation patients.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R379
本文编号：2335158
[Abstract]:Aspergillus fumigatus (Aspergillus fumigatus) is a widespread fungus in nature, which can cause severe opportunistic infection in patients with low immune function, especially in patients undergoing bone marrow transplantation or organ transplantation. Because of malignant tumor, granulocytopenia or dysfunction, and the use of glucocorticoid and immunosuppressant, the patients with immune deficiency are the high risk factors of invasive aspergillosis. In recent years, the incidence of invasive aspergillosis has been increasing and has become one of the main causes of death in immunosuppressive patients. For people with normal immune function, it can be killed and cleared by neutrophil (PMN). PMN is an important part of the human defense system and is related to the body's nonspecific anti-infection process. This kind of cells have powerful phagocytic and bactericidal function, its action process goes through chemotaxis movement, recognize and combine phagocytic substance, then ingest the intracellular, kill and degrade pathogens. However, the relationship between Aspergillus fumigatus spores and neutrophils has not been fully understood. In this study, PMN was used as the target cell, and the PMN was stimulated by Aspergillus fumigatus spores and its metabolites. The morphology of PMN, the changes of O _ 2-level, phagocytosis rate and phagocytic index of neutrophil respiratory burst were observed, respectively. To further study the pathogenesis of aspergillosis in organ transplant patients. We first used the improved method to isolate and purify the PMN, Giemsa dye from normal and liver transplantation patients. The purity of PMN was high. The proportion of PMN was 98. The living cells were measured by trypan blue rejection method. This method is simple and rapid to obtain neutrophils and has little effect on its activity. On this basis, neutrophils were incubated with Aspergillus fumigatus spores and their metabolites, and the relationship between morphological changes and phagocytosis of PMN was observed by PAS staining and electron microscopy. The morphological changes of internal and external structures of PMN were further elucidated by SEM and TEM results. We found that there were processes and pseudopods on the surface of normal PMN, granulocyte cytoplasm, homogeneous cytoplasmic density and intact mitochondrial structure. After activated by Aspergillus fumigatus spore, the surface area of PMN could increase significantly, that is, one PMN could adhere to and swallow many spores at the same time, meanwhile, during the process of endocytosis, the cellular organelles such as lysosome and endoplasmic reticulum could increase obviously. (AfD), the metabolite of Aspergillus fumigatus, has a cytotoxic effect on PMN, which may play an important role in promoting the development of Aspergillus fumigatus infection. O-2-respiratory burst is one of the main ways to perform bactericidal function after PMN activation. Flow cytometry was used to detect the level of O _ 2-produced by neutrophil respiratory burst. The results showed that the phagocytic function of PMN could be indirectly reflected by the level of O _ 2 -. The results showed that PMN was stimulated by mixed spore solution in liver transplantation patients. The respiratory burst level of O-2-was significantly lower than that stimulated by spore suspension. The changes of phagocytic function of Aspergillus fumigatus spores and AfD stimulated normal persons and liver transplantation patients were studied by PAS staining and electron microscopy. It was found that the phagocytic rate and phagocytic index of PMN were lower than those of normal subjects. The phagocytosis rate and phagocytic index of PMN stimulated by spore mixture were lower than that of spore suspension. According to the above studies, the susceptibility to Aspergillus fumigatus in liver transplantation patients is related to the lower level of respiratory burst of PMN in patients with liver transplantation, which further proves the correlation between morphological structure and function of PMN in normal persons and liver transplantation patients.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R379
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