G 试验阳性血清通过干扰 Dectin-1的内在化表达抑制 ROS 依赖性杀伤白念珠菌

G-test positive serum inhibits ROS-dependent killing of Candida albicans by interfering with internal- ized expression of neutrophil Dectin-1

目的：探讨G试验阳性血清中游离β-1，3-D-葡聚糖干扰人嗜中性粒细胞Dectin-1所介导的氧依赖性杀伤白念珠菌的机制。方法激光共聚焦显微镜检测经β-1，3-D-葡聚糖酶预处理前后的白念珠菌孢子作用下，人嗜中性粒细胞内Dectin-1表达分布的变化及与活性氧簇（ reactive oxy-gen species， ROS）的共定位关系；进一步分析Dectin-1的阻断对白念珠菌诱导下胞内ROS生成的影响；流式细胞术检测G试验阳性血清中游离的β-1，3-D-葡聚糖对白念珠菌诱导下人嗜中性粒细胞内Dectin-1和ROS表达水平的影响。结果在白念珠菌作用下，人嗜中性粒细胞内诱导性表达的Dec-tin-1主要被募集到内吞的孢子表面，且与ROS的产生具有一定的共定位关系。然而，经β-1，3-D-葡聚糖酶预处理后，胞内则未见Dectin-1的明显表达；当Dectin-1被其功能性抗体（5μg/ml）阻断后，白念珠菌诱导胞内ROS的表达与阻断前相比显著降低（LSD-t检验，P＜0．01）；经G试验阳性血清预处理的人嗜中性粒细胞中，白念珠菌诱导下胞内Dectin-1和ROS的表达水平均显著低于单一的白念珠菌作用组（LSD-t检验， P＜0．01）；线性回归分析提示G试验阳性血清中游离β-1，3-D-葡聚糖浓度分别与Dectin-1及ROS表达水平的抑制部分呈显著的剂量依存关系（ Dectin-1，R2＝0．702，P＜0．01；ROS，R2＝0．588，P＜0．01）。结论 G试验阳性血清中的游离β-1，3-D-葡聚糖可通过干扰人嗜中性粒细胞Dectin-1内在化表达的方式抑制ROS依赖性杀伤内吞的白念珠菌孢子。

Objective To investigate the mechanism of soluble β-1, 3-D-glucan in G-test positive serum in inhibiting ROS-dependent killing of Candida albicans （ C.albicans ） mediated by neutrophil Dectin-1.Methods The expression and distribution of internalized Dectin-1 and triggered ROS in human neutrophils were detected by using confocal/two-photon laser scanning microscopy upon stimulation with C.albicans （MOI=10） which was pretreated with β-1, 3-D-glucanase （10 U/ml） or not.Abrogation test was used to analyze whether intracellular Dectin-1 was involved in C.albicans-triggered ROS production in human neutrophils.Furthermore, flow cytometry analysis was performed to detect the expression of intracel-lular Dectin-1 and ROS in neutrophils which were pretreated respectively with G-test positive serum at differ-ent dilutions for 60 min and then stimulated with C.albicans for another 60 min at 37℃.Results After stimulated with C.albicans （MOI=10） for 60 min, the expression of Dectin-1 in neutrophils was recruited to the spores of opsonophagocytized C.albicans, and partly co-localized with the triggered ROS production . However, the expression of intracellular Dectin-1 was not observed in neutrophils when stimulated with β-1, 3-D-glucanase pretreated C.albicans for 60 min at 37℃.Abrogation test further showed that C.albicans-trig-gered ROS production in neutrophils was partly and irreversibly inhibited by adding Dectin -1 blocking mAb of 5 μg/ml.In addition , both the triggered expression of intracellular Dectin-1 and ROS production in neu-trophils stimulated with C.albicans （ MOI=10 ） in the presence of G-test positive serum were significantly lower than those of neutrophils stimulated only with C.albicans （LSD-t test, P〈0.01）.Linear regression a-nalysis suggested that the triggered intracellular Dectin-1 and ROS production in neutrophils upon stimulation with C.albicans were both inhibited by soluble β-1, 3-D-glucan in a dose-dependent manner （Dectin-1,R2=0.702,P〈0.01;ROS,R2=0.588,P〈0.01 ）.Conclusion Taken together, these results demonstrated that the soluble β-1, 3-D-glucan in G-test positive serum may play a role in inhibiting the ROS-dependent killing of C.albicans by interfering with internalized expression of neutrophil Dectin-1.