Rac1在小鼠中性粒细胞抗白假丝酵母菌感染中的作用

The key role of Rac1 played in mice neutrophils against Candida infection

目的评价Rac1基因在小鼠抗白假丝酵母菌感染中的作用。方法 (1)β-actin作为内参,Western blot半定量法检测两种小鼠中性粒细胞Rac1蛋白表达量;(2)趋化实验用Zigmond法经甲酰甲硫氨酰-亮氨酰-苯丙氨酸(fMLP)诱导中性粒细胞移动,延时显微镜观察中性粒细胞移动并拍照,细胞追踪软件分析小鼠中性粒细胞趋化功能;(3)fMLP诱导小鼠中性粒细胞过氧化物的产生应用异氨基苯二酰肼化学发光分析法通过微孔板发光检测仪检测过氧化物酶含量,采用细胞色素c还原法分析豆蔻酸-佛波醇-乙酸酯(PMA)引发中性粒细胞产生的过氧化物。结果假丝酵母菌耐受BALB/c小鼠中性粒细胞Rac1蛋白表达量高于假丝酵母菌易感CBA/CaH小鼠;BALB/c小鼠中性粒细胞趋化运动功能较CBA/CaH小鼠强(P<0.05);经fMLP和PMA诱导后,BALB/c小鼠中性粒细胞过氧化物的产生量高于CBA/CaH小鼠(P<0.05)。结论在小鼠中性粒细胞抗白假丝酵母菌感染过程中,Rac1能增强小鼠中性粒细胞的杀伤能力。

Objective To evaluate the effects of Rac1 gene in neutrophil during mice against Candida infection.Methods（1）Immunoblot was applied to analyze Rac1 levels in neutrophils from two strains of inbred mice with β-actin as control.（2）Neutrophils undergoing chemotaxis in response to fMLP were recorded using time-lapse imaging.Cell-tracking software was used to characterize cellular chemotaxis from captured images.（3）Neutrophil NADPH oxidase activity was measured as superoxide dismutase-inhibitable superoxide production using an isoluminol chemiluminescence assay for cells activated by fMLP or the cytochrome c reduction assay for cells activated with PMA.Results Rac1 was expressed deficiently by neutrophils from CBA/CaH mice,which also displayed significantly reduced speed compared with those from BALB/c mice in the chemotactic gradient.Furthermore,superoxide production of neutrophils from CBA/CaH stimulated with either PMA or fMLP,was lower than those from BALB/c mice.Conclusions Rac1 may play a key role for mice against-infection through mediating neutrophil chemotaxis and NADPH.