免疫抑制介导大鼠侵袭性黑曲霉菌肺病模型的构建与评价

Construction and evaluation of an immunosuppression-mediated model of invasive Aspergillus niger lung disease in rats

目的 本研究建立免疫抑制大鼠侵袭性黑曲霉菌肺病模型,为抗侵袭性肺曲霉病药物药效学评价及机制研究提供理论支持。方法 将60只SD大鼠随机分为正常对照组、环磷酰胺对照组、环磷酰胺+真菌感染低、中、高剂量组,每组12只动物。每天进行一般临床观察,分别于造模第3、7天采用ELISA法检测血清中免疫球蛋白G(IgG)、免疫球蛋白M(IgM)、血清半乳甘露聚糖(GM)水平,并检测外周血CD4^(+)、CD8^(+)细胞比例及白细胞(WBC)、中性粒细胞(Neu)含量,同时观察肺泡灌洗液黑曲霉菌负荷和大鼠肺组织形态学变化。结果 环磷酰胺对照组、环磷酰胺+真菌感染低、中、高剂量组大鼠造模后均出现自主活动减少及竖毛,且环磷酰胺+真菌感染低、中、高剂量组大鼠伴有呼吸急促,可闻及肺部湿音;与正常对照组比较,环磷酰胺对照组大鼠血中CD4^(+)、WBC、Neu、IgG、IgM水平均显著减少,CD8^(+)比例显著增加(P<0.05,P<0.01);与环磷酰胺对照组比较,环磷酰胺+真菌感染中、高剂量组大鼠血中IgG、IgM、CD4^(+)水平均显著减少(P<0.05,P<0.01),环磷酰胺+真菌感染低、中、高剂量组大鼠血中WBC、Neu水平均显著减少(P<0.05,P<0.01),环磷酰胺+真菌感染中、高剂量组大鼠血中CD8^(+)水平显著升高(P<0.05,P<0.01),环磷酰胺+真菌感染低、中、高剂量组大鼠血中GM水平及肺泡灌洗液黑曲霉菌负荷均显著增加(P<0.05,P<0.01);环磷酰胺+真菌感染低、中、高剂量组大鼠肺组织出现菌丝分布和肺泡上皮破坏、肺泡内支气管上皮杯状细胞增多、炎症细胞浸润,其病变程度与造模剂量呈正相关。结论 本研究采用黑曲霉菌联合环磷酰胺免疫抑制剂构建侵袭性黑曲霉菌肺病模型,病程与菌液浓度和造模时间呈正相关,证实细胞免疫在该病发病机制方面中发挥重要作用,同时免疫球蛋白也可影响侵袭性肺曲霉病疾病的发展过程,推测侵袭性肺曲霉病发病机制可能与体液免疫中免疫球蛋白水平有关。

Objective This study established a model of invasive Aspergillus niger lung disease in immunosuppressed rats to provide theoretical support for the pharmacodynamic evaluation of anti-invasive pulmonary aspergillosis drugs and mechanism studies.Methods Sixty SD rats were randomly divided into a normal control group;cyclophosphamide control group,and cyclophosphamide+fungal infection low,medium,and high dose groups,with 12 animals in each group.General clinical observations were performed daily,and the serum levels of immunoglobulin(Ig)G and IgM and galactomannan(GM)were detected by ELISA on the 3rd and 7th days of modeling.Simultaneously,the ratio of CD4^(+)and CD8^(+)cells,content of white blood cells(WBCs)and neutrophils(Neu)in peripheral blood,the Aspergillus niger load in alveolar lavage,and morphological changes to rat lung tissue were observed.Results Rats in the cyclophosphamide control and cyclophosphamide+fungal infection groups showed reduced voluntary activity and erect hair after modeling,and rats in the cyclophosphamide+fungal infection group also had shortness of breath and audible wet rhonchi in the lungs.Compared with the normal control group,rats in the cyclophosphamide control group showed significant reductions in the levels of CD4^(+),WBC,Neu,IgG,and IgM in the blood,and their proportion of CD8^(+)cells was significantly higher(P<0.05,P<0.01).Compared with the cyclophosphamide control group,rats in the cyclophosphamide+fungal infection medium-and high-dose groups had significantly reduced blood levels of IgG,IgM,and CD4^(+)cells(P<0.05,P<0.01);while the cyclophosphamide+fungal infection low-,medium-,and high-dose groups had significantly reduced blood levels of WBC and Neu(P<0.05,P<0.01).Additionally,rats in the cyclophosphamide+fungal infection medium-and high-dose groups had significantly increased blood CD8^(+)cells(P<0.05,P<0.01),Blood GM levels and the alveolar lavage Aspergillus niger load were significantly increased in rats in the cyclophosphamide+fungal infection low-,medium-,and high-dose groups compared with the cyclophosphamide control group(P<0.05,P<0.01).The lung tissues of the cyclophosphamide+fungal infection low-,medium-,and high-dose groups showed mycelial distribution and destruction of alveolar epithelium,increase of bronchial epithelial cup cells in the alveoli,and infiltration of inflammatory cells,and the degree of lesions was positively correlated with the modeling dose.Conclusions In this study,we used Aspergillus niger combined with cyclophosphamide immunosuppressant to construct a model of invasive Aspergillus niger lung disease.The duration of the disease was positively correlated with the concentration of bacterial fluid and modeling time,confirming that cellular immunity plays an important role in the pathogenesis of the disease.At the same time,Ig can also affect the development of invasive pulmonary aspergillosis,and it is speculated that the pathogenesis may be related to the level of Ig produced by humoral immunity.