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

中国真菌学杂志　 2014, Vol. 9 　Issue (1): 16-19.

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

王硕¹, 韩雪琳², 高欢², 宗浩¹, 韩黎²

1. 四川师范大学生命科学学院, 成都 610101;
2. 中国人民解放军军事医学科学院疾病预防控制所医院感染监控中心, 北京 100071

收稿日期:2013-11-21 出版日期:2014-02-28 发布日期:2014-02-28
通讯作者: 宗浩，E-mail：zonghao2002@yahoo.com.cn；韩黎，E-mail：Hanlicdc@163.com E-mail:zonghao2002@yahoo.com.cn;Hanlicdc@163.com
作者简介:王硕，女（汉族），硕士研究生在读.E-mail：w_s2010@126.com
基金资助:
973计划（2013CB531606），国家自然科学基金（81273229，81172801）

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

WANG Shuo¹, HAN Xue-lin², GAO Huan², ZONG Hao¹, HAN Li²

1. College of Life Science, Sichuan Normal University, Chengdu 610101, China;
2. Center for Hospital Infection Control, CDC of PLA, Beijing 100071, China

Received:2013-11-21 Online:2014-02-28 Published:2014-02-28

摘要/Abstract

摘要： 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。

关键词: 磷脂酶D, 磷脂酸, 测定方法

Abstract: 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.

Key words: phospholipase D, phosphotidic acid, detection method

中图分类号:

R379.6

王硕, 韩雪琳, 高欢, 宗浩, 韩黎. 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[J]. 中国真菌学杂志, 2014, 9(1): 16-19.

WANG Shuo, HAN Xue-lin, GAO Huan, ZONG Hao, HAN Li. 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[J]. Chinese Journal of Mycology, 2014, 9(1): 16-19.

参考文献

[1] Filler S G, Sheppard D C. Fungal invasion of normally non-phagocytic host cells[J]. PLoS pathogens, 2006, 2(12): e129.
[2] Han X, Yu R, Zhen D, et al. β-1, 3-Glucan-induced host phospholipase D activation is involved in Aspergillus fumigatus internalization into type Ⅱ human pneumocyte A549 cells[J]. PloS one, 2011, 6(7): e21468.
[3] 李平, 周汉良. 磷脂酶D和炎症的关系[J]. 生理科学进展, 1999, 30(2): 118-122.
[4] Morita S, Ueda K, Kitagawa S. Enzymatic measurement of phosphatidic acid in cultured cells[J]. J Lipid Res, 2009, 50(9): 1945-1952.
[5] Antonescu C N, Danuser G, Schmid S L. Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis[J]. Mol Biol Cell, 2010, 21(16): 2944-2952.
[6] Bligh E G, Dyer W J. A rapid method of total lipid extraction and purification[J]. Can J Biochem Physiol, 1959, 37(8): 911-917.
[7] Serrander L, Fllman M, Stendahl O. Activation of phospholipase D is an early event in integrin-mediated signalling leading to phagocytosis in human neutrophils[J]. Inflammation, 1996, 20(4): 439-450.
[8] Han X, Yu R, Ji L, et al. InlB-mediated Listeria monocytogenes internalization requires a balanced phospholipase D activity maintained through phospho-cofilin[J]. Mol Microbiol, 2011, 81(4): 860-880.
[9] Moreno-García ME, López-Bojórques LN, Zentella A, et al. CD38 signaling regulates B lymphocyte activation via a phospholipase C (PLC)-γ2-independent, protein kinase C, phosphatidylcholine-PLC, and phospholipase D-dependent signaling cascade[J]. J Immunol, 2005, 174(5): 2687-2695.
[10] Vinggaard A, Jensen T, Morgan C, et al. Didecanoyl phosphatidylcholine is a superior substrate for assaying mammalian phospholipase D[J]. Biochem J, 1996, 319 (Pt 3): 861-864.
[11] 韩丽, 毕阳, 魏晋梅, 等. 应用荧光高效液相测定麦芽根中磷脂酶 D 的活性[J]. 天然产物研究与开发, 2009, 21(2): 343-345.
[12] English D, Cui Y, Siddiqui RA. Messenger functions of phosphatidic acid[J]. Chem Phys Lipids, 1996, 80(1): 117-132.