白念珠菌磷脂酶表达水平与氟康唑耐药关系的实验研究

Relationship between phospholipase expression and fluconazole resistance in Candida albicans

目的 探讨白念珠菌毒力因子磷脂酶B1与耐药间的关系.方法 实验用白念珠菌氟康唑耐药株和敏感株各15株,提取白念珠菌总RNA,应用RT-PCR检测比较其耐药株与敏感株磷脂酶B1mRNA的表达.盐析法提取白念珠菌耐药株与敏感株胞外蛋白,P0013B放射免疫沉淀分析（RIPA）裂解液（强）裂解提取细胞内蛋白质,以β-肌动蛋白为内参照,用Western印迹法分别比较胞内和胞外耐药株和敏感株磷脂酶B1蛋白的表达水平.结果 耐药株和敏感株磷脂酶B1mRNA的相对表达量分别为0.6173±0.1090和0.2653±0.0935,耐药株磷脂酶B1mRNA的表达强于敏感株（t=2.452,P＜0.05）.耐药株和敏感株胞内磷脂酶B1蛋白相对表达量为0.4145±0.2773和0.1825±0.1831,胞外磷脂酶B1蛋白相对表达量为0.2720±0.2194和0.0688±0.0631,耐药株胞内磷脂酶B1和细胞外分泌的磷脂酶B1蛋白表达水平均高于敏感株（t=2.703、3.443,P值均＜0.05）.结论 白念珠菌耐药株毒力因子磷脂酶B1mRNA及蛋白表达水平均高于敏感株.白念珠菌磷脂酶表达水平与耐药的发生可能存在一定关系.

Objective To explore the relationship between antifungal resistance and expression of a virulence factor phospholipase B1 in Candida albicans. Methods Total RNA was extracted from 15 fluconazole-resistant and 15 fluconazole-sensitive Candida albicans isolates. RT-PCR was performed to detect the expression of phospholipase B1 mRNA. Extracellular and intracellular proteins secreted by Candida albicans cells were concentrated using salting-out method and extracted by P0013B RIPA lysis buffer, respectively followed by Western-blotting analysis to detect the expression of extracellular and intracellular phospholipase B1 protein. Results The relative expression of phospholipase B1 mRNA was 0.6173 ± 0.1090 in fluconazoleresistant Candida albicans isolates, significantly higher than in fluconazole-sensitive isolates （0.2653 ± 0.0935,P ＜ 0.05）. Increased expressions of extracellular and intracellular phospholipase B1 protein were noted in fluconazole-resistant Candida albicans isolates compared with fluconazole-sensitive isolates （0.4145 ± 0.2773 vs 0.2720 ± 0.2194, 0.1825 ± 0.1831 vs 0.2653 ± 0.0935, t = 2.703, 3.443, both P ＜ 0.05）. Conclusions The expressions of phospholipase B1 mRNA and protein are elevated in fluconazole-resistant Candida albicans isolates, suggesting that the expression of phospholipase B1 may be associated with the occurrence of antifungal resistance in Candida albicans.