磷脂酶D缺失对小鼠抗烟曲霉感染过程的影响研究

Loss of phospholipase D in mice against Aspergillus fumigatus infection

目的揭示宿主磷脂酶D(phospholipase D,PLD)应对烟曲霉感染过程中的重要作用和可能机制。方法应用滴鼻方式使野生小鼠和磷脂酶D双基因敲除小鼠(pld1^(-/-)pld2^(-/-))感染烟曲霉孢子后,取小鼠肺组织、抽取支气管肺泡灌洗液,应用组织匀桨与菌落计数的方法检测小鼠肺组织中烟曲霉负荷,采用eBioscience公司的多因子检测试剂盒检测肺泡灌洗液中炎症因子分泌情况;分离两种小鼠的骨髓细胞并诱导分化为成熟巨噬细胞(BMDM),制霉菌素法检测其吞噬和杀死烟曲霉孢子的能力。结果感染烟曲霉孢子6 h后,pld1^(-/-)pld2^(-/-)小鼠肺部真菌负荷显著高于野生小鼠,其肺泡灌洗液和肺泡巨噬细胞中均存在大量孢子,肺泡灌洗液中炎症因子浓度显著升高;体外实验证明pld1^(-/-)pld2^(-/-)小鼠的BMDM细胞吞噬和杀死烟曲霉孢子的能力显著减弱。结论小鼠pld1基因和pld2基因同时敲除导致其巨噬细胞的吞噬和杀死烟曲霉孢子的能力显著减弱,进而可能降低小鼠抗烟曲霉感染的能力。同时机体可能通过分泌大量的炎症因子以招募其他免疫细胞杀灭烟曲霉孢子。

Objective To reveal the important role and the possible mechanism of the host phospholipase D in response to the infection of Aspergillus fumigatus.Methods Wild type mice and phospholipase D double genes knockout mice（pld1^-/-pld2^-/-）were infected with Aspergillus fumigatus conidia by nasal instillation.After infection,the lung tissure burden of Aspergillus fumigatus was detected by tissue homogenization and CFU counting.The secretion of inflammatory factors in BALF were detected by multi factor detection kit from eBioscience Company.Bone marrow cells of two kinds of mice were isolated and differentiated into mature macrophages（BMDM）,their ability to phagocyte and kill Aspergillus fumigatus was detected by the nystatin protection method.Results Six hours post infection,the load of pulmonary fungi in pld1^-/-pld2^-/-mice was significantly higher than that of wild type group;and a large number of Aspergillus fumigatus spores were found in BALF and alveolar macrophages of pld1^-/-pld2^-/-mice.Meanwhile,the concentration of inflammatory factors in BALF of pld1^-/-pld2^-/-mice was significantly higher.In vitro experiments showed that the phagocytosis and killing ability of BMDM of pld1^-/-pld2^-/-mice was significantly reduced.Conclusion The simultaneous knockout of the pld1 and pld2 genes in mice significantly decreased the phagocytosis and fungicidal ablities of the macrophage,which might interfere mice againsting A.fumigatus infection.Meanwhile,the pld1^-/-pld2^-/-mice might secrete a large number of inflammatory factors to recruit other immune cells to kill Aspergillus fumigatus conidia.