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目的建立侵袭性肺曲霉菌病模型,探讨不同免疫状态对小鼠抵抗曲霉菌感染能力的影响。方法2006年1月至12月于中国上海生命科学院生物细胞所通过腹腔内注射环磷酰胺后鼻腔内滴入烟曲霉菌(Af)孢子制备侵袭性肺曲霉菌病小鼠模型;获取小鼠肺组织并进行匀浆,接种培养后进行菌落计数,酶联免疫吸附试验(ELISA)法检测γ-干扰素(IFN-γ)质量浓度,部分肺组织进行苏木精-伊红(HE)和六胺银(GMS)染色;以反转录-聚合酶链反应(RT-PCR)检测小鼠肺脏IFN-γ的mRNA表达,观察小鼠死亡率。结果免疫抑制小鼠接种烟曲霉菌后萎靡少动,呼吸急促,口周围出血严重,很少进食水,多随时间延长而表现恶化,肺部真菌负荷呈明显增加趋势,支气管肺泡灌洗液(BALF)中IFN-γ质量浓度随时间推移逐渐升高,48h后保持于较高水平[(932.6±208.36)ng/L],约50%小鼠3d内死亡。病理显示对照组小鼠于接种烟曲霉菌后,支气管旁有大量多型核白细胞(PMN)聚集,炎症呈灶性分布,肺泡壁增厚。免疫受损(ICH)组接种烟曲霉菌后,炎症弥漫分布,肺泡及间质水肿出血剧烈,烟曲霉菌孢子、菌丝呈灶状分布,至48h出现支气管壁破坏、支气管周围坏死、大量炎症细胞浸润,烟曲霉菌菌丝集落成簇出现。结论肺部细胞因子IFN-γ质量浓度与曲霉菌负荷呈一定相关性,机体的免疫功能、T细胞介导的免疫在拮抗曲霉菌感染中发挥着重要作用。
Objective To establish an invasive pulmonary aspergillosis model and investigate the effect of different immune status on the ability of mice to resist Aspergillus infection. Methods The mouse model of invasive pulmonary aspergillosis was prepared by intranasal instillation of Aspergillus fumigatus (Af) spores after intraperitoneal injection of cyclophosphamide in biological cells from Shanghai Institutes of Biological Sciences, China from January to December 2006. (HE) and six (6) HEK293 cells were harvested and cultured. The colonies were counted after inoculation and culture. The mass concentration of IFN-γ was detected by enzyme linked immunosorbent assay (ELISA) (GMS) staining. The mRNA expression of IFN-γ was detected by reverse transcription-polymerase chain reaction (RT-PCR). The mortality of mice was observed. Results Immunosuppressed mice inoculated with Aspergillus fumigatus showed less apomictism, shortness of breath, severe bleeding around the mouth, little water intake, more deterioration with time, and a significant increase in the lung fungal load. The bronchoalveolar lavage fluid ( The concentration of IFN-γ in BALF increased gradually with the passage of time and maintained at a high level after 48h [(932.6 ± 208.36) ng / L]. About 50% of mice died within 3 days. Pathological examination showed that after inoculation with Aspergillus fumigatus, a large number of polymorphonuclear leukocytes (PMN) accumulated in the bronchial epithelium of mice in the control group, with focal distribution of inflammation and thickening of the alveolar wall. Immunofluorescence (ICH) group inoculated with Aspergillus fumigatus, diffuse distribution of inflammation, severe alveolar and interstitial edema bleeding, Aspergillus fumigatus spores, mycelium was focal distribution, bronchial wall destruction occurred 48h, bronchial necrosis, a large number of inflammation Cell infiltration, Aspergillus fumigatus mycelium clusters appear. Conclusion The concentration of cytokines IFN-γ in the lung is related to the aspergillus load. The immune function and T cell-mediated immunity plays an important role in antagonizing Aspergillus infection.