白色念珠菌烯醇化酶诱导产生中性粒细胞外网

Recombinant enolase of Candida albicans could induce the formation of neutrophil extracellular traps

目的通过重组白色念珠菌烯醇化酶(enolase),研究其对人中性粒细胞的作用。方法采用分子克隆法,构建质粒、克隆、表达、纯化白色念珠菌重组enolase,密度梯度离心法提取人中性粒细胞,通过2,7-二氯二氢荧光素二醋酸酯(DCFH-DA)法、Sytox green染色和免疫荧光观察白色念珠菌重组enolase(500 nmol/L)对人中性粒细胞的刺激作用。中性粒细胞加入白色念珠菌SC5314(中性粒细胞∶白色念珠菌=5∶1)作为阳性对照组(SC5314组),未加刺激的作为阴性对照组。采用单因素方差分析(One-Way ANOVA)及Tukey′s检验对数据进行统计分析。结果成功得到重组enolase,分子量为46 kDa。重组enolase可以刺激人中性粒细胞产生细胞内活性氧(ROS),enolase组与SC5314组产生ROS的量均无明显差异。Sytox green对胞外DNA定量及免疫荧光观察,均显示重组enolase可以刺激人中性粒细胞产生中性粒细胞外网(NETs)。NETs定量结果显示,enolase组[(11.0±2.5)%]与SC5314组[(12.4±2.0)%]在2 h时无明显差异(F=6.13,P=0.0886),在4 h时enolase组[(22.2±2.0)%]显著低于SC5314组[(31.4±3.1)%],差异有统计学意义(F=9.745,P=0.0370)。结论白色念珠菌enolase可以诱导人中性粒细胞形成NETs。

Objective To investigate the effect of enolase of Candida albicans(C.albicans)onhuman neutrophils. Methods Molecular cloning method was applied to plasmids construcion,cloning,expression and recombinant enolase purification,and density gradient centrifugation was used to isolatethe human neutrophils. The effects of recombinant enolase on human neutrophils were observed byDCFH-DA method,SYTOX green and immunofluorescence. The positive control(SC5314 group)wasneutrophils stimulated by C.albicans SC5314(neitrophil∶C.albicans = 5∶1)and the negative control wasneutrophils without stimulation. One-Way ANOVA and Tukey′s test were applied for statistical analysis.Results Recombinant enolase,46 kDa,was successfully cloned and purified. Recombinant enolase couldstimulate neutrophils to produce the intracellular ROS,which has no significant difference compared withthose induced by C.albicans SC5314 group at 30,60 and 120 min. Sytox green quantitative and immunoflu-orescence observation of extracellular DNA showed that recombinant enolase also could induce humanneutrophils to form NETs. NETs quantitative results showed that there was no significant differencebetween SC5314 group[(12.41 ± 2.00)%]and enolase group[(11.0 ± 2.5)%]at 2 h(F = 6.13,P =0.0886),but enolase group[(22.2 ± 2.0)%)]was significant lower than SC5314 group[(31.4 ± 3.1)%]at 4 h(F = 9.745,P = 0.0370). Conclusion Recombinant enolase of C.albicans could induce human neu-trophils to form NETs.