核素掺入研究氟康唑对真菌麦角甾醇生物合成的影响

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10 min后,加[1-14C]乙酸钠振荡培养3 h,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14C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,14C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,14C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32.17 nmol/L.结论:核素掺入法能定量考察羊毛甾醇14α-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,为其他抗真菌药物的作用机制和抑酶活性研究提供了新方法.

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

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.