HLA-A*0201转基因小鼠侵袭性肺曲霉病模型的建立与鉴定

Establishment and Identification of an Invasive Pulmonary Aspergillosis Mouse Model in HLA-A*0201 Transgenic Mice

【目的】建立HLA-A*0201转基因小鼠侵袭性肺曲霉病模型,并进行实验室鉴定。【方法】在真菌孢子感染前,给予HLA-A*0201转基因小鼠腹腔注射环磷酰胺。乙醚麻醉后,小鼠经鼻腔吸入3组不同浓度的(3×106、3×107、3×108mL-1)烟曲霉孢子悬液。阴性对照组小鼠吸入PBS。感染后36 h,处死各组小鼠,取肺组织病理行HE、PAS以及GMS染色分析,以验证感染是否成功并计算肺侵袭性肺烟曲霉感染小鼠模型成模率。肺组织研磨涂皿后行菌落计数,分析小鼠肺烟曲霉负载量。【结果】小鼠肺组织病理检查显示,正常肺组织结构遭到显著破坏,大量菌丝生长并可见菌丝侵犯血管现象,证实侵袭性肺曲霉病模型造模成功。在免疫抑制的方法和条件恒定时,3种孢子浓度由低到高成模率分别为36.7%、76.7%、96.7%。各实验组成模小鼠肺组织研磨液在PDA培养基上均有大量烟曲霉菌落生长,未感染小鼠组未见烟曲霉菌落生长。【结论】成功建立HLA-A*0201转基因小鼠侵袭性肺曲霉病模型,在免疫抑制的方法和条件恒定时,模型成模率与接种孢子悬液的浓度成正比,采用高浓度的孢子悬液(3×108mL-1)时,可以达到较高的成模率。

[Objective] To Establish and identify an invasive pulmonary aspergillosis mouse model in HLA-A ＊ 0201 transgenic mice. [Methods] The HLA-A ＊ 0201 transgenic mice were treated with cyclophosphamide before challenge with fungal conidia. Aspergillusfumigatus （BMU 01200, provided by Research Center for Medical Mycology and Mycoses, Department of Dermatology and Venerology, First Hospital, Peking University） conidia suspension of various concentrations （3 × 10^5/mL, 3 × 10^7/mL, 3 × 10^8/ mL） were intranasal dripped right after the mice were anesthetized by diethyl ether. The mice administrated with PBS were employed as negative controls. 36 h post infection, the mice of different groups were sacrificed and lung tissue histopathology analysis with HE, PAS and GMS staining were performed. Colony forming unit （CFU） counting was also executed to confirm the infection. [ Resuhs ] Histopathology analysis characterized by lung tissue destruction and hyphal invasion demonstrated the invasive pulmonary Aspergillus fumigatus infection in the mice model. The invasive pulmonary Aspergillus fumigatus infection of mice was also identified by CFU counting of grind lung tissue in four experimental mice groups, the success rate of which O, 36.7%, 76.7% and 96.7%, respectively. [Conclusions] We successfully established an invasive pulmonary aspergillosis mouse model in HLA-A ＊ 0201 transgenic mice, with the highest success rate of 96.7% in the group of the highest conidia dose administrated. This model could be utilized to investigate the immunotherapy effect of human HLA-A ＊ 0201 restricted AspergiUus fumigatus antigen peptide specific T lymphocytes against Aspergillus fumigatus in vivo.