磷脂酸含量测定法评价烟曲霉孢子对肺上皮细胞磷脂酶D活性的影响

The use of a method for determining the levels of cellular phosphatidic acid in detecting the Aspergillus fumigatus activating phospholipase D activity in pulmonary epithelial cells

目的 优化检测烟曲霉刺激A549细胞后磷脂酸（phosphotidic acid,PA）含量变化的方法,间接反应细胞内磷脂酶D（Phospholipase D,PLD）活性变化.方法 建立烟曲霉ATCC13073刺激肺上皮细胞模型;采用甲醇氯仿法提取胞内脂质;用改良的磷脂酸含量测定法测定PA标准品和细胞内PA水平变化规律.结果 PA标准品在5～ 250 μmol/L呈线性关系;经膨胀孢子刺激后,肺上皮细胞内PA含量显著升高,休眠孢子在这一过程中对肺上皮细胞内PA含量无明显作用.结论 改良的PA测量法能快速、稳定而有效地测定细胞内的PLD活性.烟曲霉膨胀孢子能显著激活肺上皮细胞内的PLD活性.

Objective To establish a method to detect Phospholipase D （PLD） activity in the A549 cells stimulated by A.fumigatus by measuring the levels of cellular phosphatidic acid （PA）.Method Using the interaction model of epithelial cells stimulated by A.fumigatus ATCC13073 ;Chloroform-methanol method was used to extrat the intracellular lipid.A modified method was established to measure PA standard and the levels of cellular PA.Results The calibration curve of PA standard was linear between 5 and 250 μmol/L.Swollen conidia,but not resting conidia increased the level of PA in A549 cells.Conclusion This improved method for determining the levels of cellular PA was high sensitive,stable and convenient for measuring indirectly the intracellular PLD activity.Our data indicated that the PLD activity in the pulmonary epithelial cells could be significantly activated by A.fumigatus swollen conidia.