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。烟曲霉孢子被患者吸入后,可以从气管肺泡中侵入到肺部造成初发的侵袭性感染,并常伴远隔器官的转移。近10 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,它可以被肺泡巨噬细胞(PAM)杀灭并清除。然而烟曲霉孢子与肺泡巨噬细胞之间的关系尚未阐明。
PAM 在肺组织感染的防御中起着十分重要的作用。当它被外来刺激物激活时,细胞外信号可通过与受体结合,激活信号级联通路并传递到达细胞核,激活或失活核转录因子,作用于目的基因的调控序列而引起相应蛋白质表达谱的改变。从而既提高自身吞噬、处理、递呈抗原的能力,又分泌多种细胞因子和化学趋化因子,如白介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、巨噬细胞炎症蛋白(MIP)以及白三烯B4(LTB4)等,促进T、B 细胞的增殖、成熟,募集中性粒细胞至肺,全面提高肺的免疫能力以抵御外来刺激物的不良作用。实验证明,这些炎症因子在抗烟曲霉感染中起着重要作用。NF-κB 是广泛存在于真核生物中,一种由复杂的多肽亚单位组成的蛋白家族。它作为信号传导途径中的枢纽,与免疫、肿瘤的发生、发展,细胞凋亡的调节以及胚胎发育等重要事件有着密切联系,是一种重要的核转录因子。研究发现,在烟曲霉感染中,PAM 的NF-κB 信号转导通路的激活可能起着重要作用。
本实验以PAM 为研究靶细胞,经烟曲霉孢子及其代谢产物的刺激,分别观察PAM的形态学改变、肺泡巨噬细胞炎症因子的释放水平的改变以及相关信号通路的研究,为进一步探讨肺烟曲霉病的发病机制的研究奠定基础。
我们首先建立并稳定了正常PAM 和免疫抑制PAM 体外培养模型,经瑞氏染色和巨噬细胞的标记抗原CD68 行免疫组化试验对巨噬细胞进行了鉴定。在此基础上,将肺泡巨噬细胞与烟曲霉孢子及其代谢产物共孵育,通过扫描电镜和透射电镜观察PAM形态学变化。我们发现,正常PAM 表面有大量微皱褶或微皱襞,在被烟曲霉孢子激活后,PAM 可以明显增大细胞的表面积,即可以使一个巨噬细胞同时粘附和吞噬多个
Aspergillus is a kind of fungus capable of causing life-threatening opportunistic infections in immunocompromised patients. Patients with host defenses compromised by malignancy, granulocytopenia, neutrophil dysfunction, corticosteroid therapy, or immunosuppressive drugs are at great risk. Inhalation of airborne Aspergillus conidia in the alveoli primarily results in pulmonary infection, usually with dissemination to other organs. The incidence of invasive aspergillosis (IA) has increased considerably in the past decade, and this infection is a major cause of mortality in immunocompromised hosts. To a nomal immune function man, Aspergillus conidia can be cleared off by pulmonary alveolar macrophage (PAM).Unfortunately, the relarionship between PAM and Aspergillus conidia is still unclear.
PAM is very important in the protection of lung infection. When it is activated by exotic matierials, the exotic cellular signals can change the protein mass spectra by activating or inactivating transcription factors, regulate-controlling sequences. It can not only boost the abilities of phagotosis, disposal and presentation of antigens but also excrete multiple cytokines and chemotaxis mediators such as interleukin 1β(IL-1β), tumor necrosis factor α(TNF-α), macrophage inflammatory protein(MIP) and leukotriene B4 (LTB4) , et al, promote T and B cells to proliferate and mature, collect neutrophil to lung, raise the immune ability of lung to resist the bad effect from exotic materials. It has already been confirmed by experiments that those inflammatory mediaters are very important factors in the protection of Aspergillus infection. NF-κB, a protein family consisting of complex sub-polypeptide units, exists ubiquitouly in Eukaryota. NF-κB is a central cell nuclear transcription factor in signal conducting pathway, relating with many important funtions such as immune, the occuring and developping of tumor, the regulating of cell units and embryo growth, et al. It is reported that the activation of NF-κB is very important during the Aspergullus infection.
In our experiment, using PAM as target cells, we observed the morphologic changes, the releasing levels of inflammatory mediators of PAM and relating signal pathway research when stimulated by Aspergillus fumigatus conidia and its metalites. Firstly, we established and stablized the models of PAM and immunosupressive PAM in vitro, PAM was identified with Reiz coloration and immune histochemistry methods. Then we used scan and transmiss electron microscopy to observe the morphologic changes by incubating PAM and Aspergillus fumigatus conidia or metablites together or alone. The results suggest that exterior of nomal PAM has plenty of microruga, the surface area of PAM was increased so as to make it possible to conglutinate and englut more conidia when activated by Aspergillus fumigatus conidia; at the same time, it provides the histologic evidence of augmentation of lysosome and endoplasmic reticulum. Aspergillus fumigatus’s metablites such as gliotoxin and Aspergillus fumigatus diffusate(AfD) have some cellular toxic functions on PAM and may do accelerating functions in the development of Aspergillus infection. Gliotoxin can induce the apotosis of PAM. We haven’t found any toxic function of fumagillin and helvelic acid on PAM.The incidence of PAM rise greatly when the quantity of Aspergillus fumigatus conidia increased. Secondly, we checked up the levels of cytokines(IL-1βand TNF-α) in the culture supernatant by ELISA. We found that Aspergillus fumigatus conidia can stimulate nomal PAM to release much IL-1βand TNF-α, having quantity-effect and time-effect relationship. Immunosuppresive PAM releases less IL-1βand TNF-αthan nomal PAM does. AfD can inhibit the releasing of IL-1βand TNF-αpartly stimulated by Aspergillus fumigatus conidia. The results suggest that AfD do some toxic functions on PAM and may accelerate the development of Aspergillus infection. Because the well known materils of toxic Aspergillus fumigatus do not have obvious effect on PAM, it is necessary to make more research on the component of AfD. There are three methods to study transcription factors: electrophoretic mobility shift assay(EMSA), western blot(WB) and gene analysis, but these methods just can do semi-quantity, spend too much time and are lack of specificity and sensitivity. ELISA of checking NF-κB is a new method to check its energy. It can be more easily and spend less time to check up NF-κB quantificationally. Research had suggested that TLR may do some functions in the interaction between Aspergillus fumigatus conidia and PAM. In our
research, we used anti-TLR4 to block the TLR4 on the membrane of PAM and then observed the pathway activated by Aspergillus fumigatus conidia from cellular view. We confirmed that Aspergillus fumigatus conidia could activate PAM, improve the levels of IL-1βand TNF-αreleased by PAM which relating with activated of NF-κB, but its function could be blocked partly by TLR4(the cytokine turned less when TLR4 had been blocked ). Based on above, we conclude that the interaction beween Aspergillus fumigatus conidia and PAM is associated greatly with the activation of NF-κB. From our experiments, we confirm some relativity between the morphologic results and its functions. At the same time, Aspergillus fumigatus conidia could activate nomal PAM and stimulate it to release high levels of IL-1βand TNF-α, while immunosupressive PAM release less IL-1βand TNF-α, which suggest that high levels of IL-1βand TNF-αare in favor of the cleatance of Aspergillus fumigatus conidia. Morever, NF-κB is a central factor in PAM when it is activated by Aspergillus fumigatus conidia, but other signal pathway may participate in this activation of PAM.
引文
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