煙曲霉對(duì)人中性粒細(xì)胞結(jié)構(gòu)和功能的影響
發(fā)布時(shí)間：2018-11-16 09:37
【摘要】： 煙曲霉是自然界廣泛存在的真菌,可以引起免疫功能低下的病人,尤其是進(jìn)行了骨髓移植或器官移植的病人嚴(yán)重的機(jī)會(huì)感染。因?yàn)閻盒阅[瘤、粒細(xì)胞減少或功能障礙、糖皮質(zhì)類固醇和免疫抑制劑的使用等原因造成的患者免疫功能缺陷,是侵襲性曲霉病的高危因素。近年來(lái),侵襲性曲霉病發(fā)病率不斷上升,并成為免疫抑制患者死亡的主要原因之一。對(duì)于免疫功能正常的人來(lái)說(shuō),它可以被中性粒細(xì)胞(PMN)殺滅并清除。PMN是人體防御系統(tǒng)的重要組成部分,與機(jī)體的非特異性抗感染過(guò)程有關(guān),該類細(xì)胞具有強(qiáng)大的吞噬殺菌功能,其作用過(guò)程歷經(jīng)趨化運(yùn)動(dòng),識(shí)別并結(jié)合吞噬物質(zhì),進(jìn)而攝入胞內(nèi),最終殺傷并降解病原體。然而煙曲霉孢子與中性粒細(xì)胞之間的關(guān)系尚未完全清楚。 本實(shí)驗(yàn)以PMN為研究靶細(xì)胞,主要以煙曲霉孢子及其代謝產(chǎn)物對(duì)它的刺激,分別觀察PMN的形態(tài)學(xué)、中性粒細(xì)胞呼吸爆發(fā)產(chǎn)生O2-水平以及吞噬率和吞噬指數(shù)的改變,為進(jìn)一步探討器官移植病人易患煙曲霉病的發(fā)病機(jī)制的研究奠定基礎(chǔ)。 我們首先用改良法分離和純化正常人和肝移植患者的PMN,姬姆薩染液示PMN的純度較高,其比例98%,臺(tái)盼藍(lán)排斥法測(cè)活細(xì)胞98%。此法能夠既簡(jiǎn)單又快速獲得中性粒細(xì)胞且對(duì)其活性影響較小。在此基礎(chǔ)上,將中性粒細(xì)胞與煙曲霉孢子及其代謝產(chǎn)物共孵育,利用PAS染色和電鏡技術(shù)觀察PMN的形態(tài)學(xué)改變與吞噬功能的關(guān)系;通過(guò)掃描電鏡和透射電鏡結(jié)果進(jìn)一步闡明PMN內(nèi)部及外部結(jié)構(gòu)的形態(tài)學(xué)變化。我們發(fā)現(xiàn),正常PMN表面有突起及偽足形成,粒細(xì)胞胞漿、胞質(zhì)密度均勻,線粒體結(jié)構(gòu)完整。在被煙曲霉孢子激活后,PMN的表面積可以明顯增大,即可以使一個(gè)PMN同時(shí)粘附和吞噬多個(gè)孢子;同時(shí),在細(xì)胞內(nèi)吞過(guò)程中,細(xì)胞內(nèi)溶酶體和內(nèi)質(zhì)網(wǎng)等細(xì)胞器可明顯增多。煙曲霉的代謝產(chǎn)物----煙曲霉?jié)B出物(AfD)對(duì)PMN存在一定細(xì)胞毒性作用,這在促進(jìn)煙曲霉感染的發(fā)展中可能起著重要的作用。 O2-呼吸爆發(fā)是PMN激活后行使殺菌功能的主要途徑之一。運(yùn)用流式細(xì)胞儀檢測(cè)中性粒細(xì)胞呼吸爆發(fā)產(chǎn)生O2-水平可以間接地反映PMN的吞噬功能,結(jié)果顯示肝移植患者PMN經(jīng)孢子混合液刺激后,其O2-的呼吸爆發(fā)水平明顯低于經(jīng)孢子懸液刺激的結(jié)果。通過(guò)煙曲霉孢子及AfD刺激正常人和肝移植患者PMN,利用PAS染色和電鏡技術(shù)對(duì)其吞噬功能的變化進(jìn)行研究發(fā)現(xiàn)肝移植患者PMN吞噬煙曲霉孢子的吞噬率和吞噬指數(shù)低于正常人;患者PMN經(jīng)孢子混合液刺激后,其吞噬率和吞噬指數(shù)低于經(jīng)孢子混懸液刺激的結(jié)果。 通過(guò)以上研究,肝移植患者易感煙曲霉菌與其PMN的呼吸爆發(fā)水平較低有關(guān),也進(jìn)一步證明了正常人和肝移植患者PMN的形態(tài)學(xué)結(jié)構(gòu)和其功能的相關(guān)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R379
本文編號(hào)：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R379
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