核素掺入研究氟康唑对真菌麦角甾醇生物合成的影响  

Effect of fluconazole on ergosterol biosynthesis in fungi by measuring incorporation of nuclide

作　　者：曹永兵[1] 高平挥[1] 张军东[1] 徐铮[1] 王彦[1] 贾鑫明[1] 张万年 姜远英[1] 

机构地区：[1]第二军医大学药学院药理学教研室,上海200433 [2]第二军医大学药学院药物化学教研室

出　　处：《第二军医大学学报》2004年第7期751-754,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金 ( 3 0 2 0 0 0 12 ) ;上海市科技发展基金( 0 2 QMA14 0 8) .

摘　　要：目的 :用核素掺入结合放射自显影和液体闪烁计数方法 ,研究抗真菌药物氟康唑对真菌麦角甾醇生物合成的影响。方法 :取新鲜培养的新生隐球菌标准株分别与不同浓度氟康唑预培养 1 0 min后 ,加 [1 - 1 4C]乙酸钠振荡培养 3h,经皂化、提取、薄层展开 ,使角鲨烯、羊毛甾醇和麦角甾醇等分离后 ,用液体闪烁计数法测定上述各成分中 1 4C的掺入量 ,并计算药物对真菌麦角甾醇生物合成的半数抑制浓度 (IC50 )。结果 :在空白对照组中 ,1 4C主要掺入麦角甾醇 ,而经氟康唑作用后 ,真菌的麦角甾醇生物合成受阻 ,1 4C在麦角甾醇中的掺入减少 ,但在羊毛甾醇及其上游成分角鲨烯环氧化物中的积累增多 ,其含量变化均呈剂量依赖性。氟康唑对真菌麦角甾醇生物合成的 IC50 为 32 .1 7nmol/L。结论 :核素掺入法能定量考察羊毛甾醇 1 4 α-去甲基化酶抑制剂氟康唑对羊毛甾醇去甲基化反应的抑制作用 。Objective:To study the effect of fluconazole on ergosterol biosynthesis in fungi by measuring the incorpora-tion of acetate.Methods:Saponification and extraction of the nonsaponifiable lipids(NSLs)of Cryptococcus neofor-mans were carried out after3h of incubation with acetate and different concentrations of fluconazole.Radioactive com-ponents of NSLs were separated by thin-layer chromatography and located by autoradiography.Radioactive zones were cut from plate and assayed for radioactivity by liquid scintillation counting.Results:Fluconazole inhibited the incorporation of acetate into ergosterol and increased the incorporation of acetate into lanosterol and oxidosqualene in a dose-de-pendent manner in fungi.Conclusion:Measuring the incorporation of acetate into NSLs of fungi can evaluate the in-hibitory effect of fluconazole on ergosterol biosynthesis,which may be widely applied to studies on mechanism of antifungal a-gents.

关 键 词：氟康唑 羊毛甾醇14α-去甲基化酶 麦角甾醇 羊毛甾醇 核素掺入 

分 类 号：R978.5[医药卫生—药品]