申克孢子丝菌酵母相与白念珠菌诱导中性粒细胞活性氧簇的差异性表达

Differential expression of reactive oxygen species in human neutrophils stimulated by the yeast phase of Sporothrix Schenckii and Candida albicans

目的研究人中性粒细胞吞噬申克孢子丝菌酵母相和白念珠菌后胞内活性氧簇（ROS）的实时表达水平及对两者杀菌能力的差异。方法应用密度梯度离心法分离人外周血中性粒细胞，并通过ROS探针（DCFH—DA）、流式细胞仪和激光共聚焦显微镜检测申克孢子丝菌酵母相临床株和白念珠菌标准株ATCC90028作用后，中性粒细胞内ROS的实时表达水平及对上述2种真菌孢子的杀菌率。结果申克孢子丝菌酵母相临床株和白念珠菌标准株ATCC90028作用中性粒细胞60min后，申克孢子丝菌组中性粒细胞内ROS表达水平达到峰值（平均荧光强度159．67±11．34），并显著高于白念珠菌组（112．22±9．66），经LSD—t检验，P〈0．01；而作用120～180min后，胞内ROS表达水平（120min为89．01±9．81；180min为57．63±8．46）迅速下降，显著低于白念珠菌组中ROS的同期表达水平（120min时为110．25±7．28；180min时为109．98±9．00，经LSD—t检验，120min时P〈0．05，180min时P〈0．01）。激光共聚焦显微镜观察到，ROS主要表达于吞噬上述真菌孢子的中性粒细胞中，并被募集到被吞噬的部分孢子表面。中性粒细胞对申克孢子丝菌180min杀菌率（19．21％±3．68％）亦显著低于其对白念珠菌孢子的杀菌率（26．63％±4．97％），经LSD—t检验，P〈0．01。结论中性粒细胞吞噬上述2种真菌孢子后，胞内ROS呈明显的差异性表达。与白念珠菌相比，中性粒细胞内ROS水平的迅速下降在一定程度上抑制了其对申克孢子丝菌的杀伤效能。

Objective To compare the reactive oxygen species （ROS） expression in human neutrophils phagocytizing Sporothdx Schenckii and Candida albicans yeast cells, and to compare the fungicidal activity of human neutrophils against Spomthffx Schenckii and Candida albicans. Methods Human neutrophils were isolated from peripheral blood by using density gradient centrifugation method, and cultured with the presence of the yeast phase of a Sporothffx Schenckii clinical isolate and a standard strain of Candida albicans （ATCC 90028） at a multiplicity of infection of 10 or 1 for 60 - 210 minutes. Subsequently, flow cytometry with ROS probe （2＇,7＇- dichlorofluorescein diacetate, DCFH-DA） was carried out to for the real time detection of intracellular ROS level, confocal laser scanning microscopy （CLSM） for the observation of ROS distribution. In addition, the fungicidal efficiency of neutrophils against Sporothrix Schenckii and Candida albicans was estimated by the number of colonies after additional culture of neutrophil lysates on brain-heart infusion agar （BHIA） medium. Statistical analysis was done by using univariate analysis of variance and LSD-t test. Results The intracellular ROS level peaked at 60 minutes in neutrophils incubated with Sporothnx Schenckii yeast cells, then decreased rapidly from 60 minutes to 210 minutes. Compared with the neutrophils incubated with Candida albicans yeast cells, those with Sporothdx Schenckii yeast cells showed a higher ROS level （expressed as mean fluorescence intensity） at 60 minutes （159.67 ± 11.34 vs. 112.22 ± 9.66, P 〈 0.01）, but a lower ROS level at 120 minutes （89.01 ±9.81 vs. 110.25±7.28, P 〈 0.05） and 180 minutes （57.63 ±8.46 vs. 109.98± 9.00, P 〈 O.01）. CLSM revealed that ROS was mainly distributed in neutrophils with phagocytized fungal spores, and especially on the surface of phagocytized spores. Furthermore, the percentage of yeast cells killed by neutrophils was significantly lower for Sporothnx Schenckii than for Candida albicans at 180 minutes （ 19.21% ± 3.68% vs. 26.63% ± 4.97%, P 〈 0.01 ）. Conclusions Differential expression of intracellular ROS was observed in neutrophils after phagocytosis ofCandida albicans and Spomthrix schenckii. Neutrophils exert a stronger fungicidal activity against Sporothrix Schenckii in comparison with Candida albicans, which may be associated with the rapid decrease of ROS level in neutrophils after phagocytosis.