色谱

色谱 ›› 2011, Vol. 29 ›› Issue (04): 330-334.DOI: 10.3724/SP.J.1123.2011.00330

• 特别策划 • 上一篇    下一篇

氨基酸消耗谱在区分不同作用机制抗癌药物中的应用

韩晓菲, 王龙星, 杨乾栩, 肖红斌*   

  1. 中国科学院大连化学物理研究所, 辽宁 大连 116023
  • 收稿日期:2011-03-15 修回日期:2011-03-21 出版日期:2011-04-28 发布日期:2011-04-28
  • 通讯作者: 肖红斌,博士,研究员,主要研究方向为中药药效物质基础及作用机理. Tel: (0411)84379756, E-mail: hbxiao@dicp.ac.cn.
  • 基金资助:
    国家自然科学基金项目(No. 20705033).

Classification of anticancer drugs with different mechanisms based on amino-acid consumption profiling in culture media

HAN Xiaofei, WANG Longxing, YANG Qianxu, XIAO Hongbin*   

  1. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
  • Received:2011-03-15 Revised:2011-03-21 Online:2011-04-28 Published:2011-04-28

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摘要： 以邻苯二甲醛为衍生试剂,正缬氨酸为内标,采用柱前在线衍生高效液相色谱(HPLC)分离,荧光检测(FLD)的方法,建立了培养基中游离氨基酸(FAA)代谢消耗谱的定量分析方法。流动相A液为10 mmol/L Na2HPO4-Na2B4O7缓冲液(pH 7.95), B液为乙腈-甲醇-水(45:45:10, v/v/v);二元线性梯度洗脱,流动相B在35 min内由5%升至52%,33 min内17种FAA全部得到良好分离。应用该方法分别测定了HeLa细胞在紫杉醇和丝裂霉素干预24 h后的培养基氨基酸代谢消耗谱(以正常培养组为对照)。以相对峰面积作为原始数据,通过Matlab7.1软件平台,用偏最小二乘判别分析(PLS-DA)得分图进行关联研究,结果表明: 与对照组相比,培养基游离氨基酸代谢消耗谱能够区分两种不同机制的抗癌药物,为抗癌药物筛选过程中的药物作用机制预归类提供了一种新的技术方法。同时,这种以外观内的方法具有方便、经济的特点。

关键词: HeLa细胞, 氨基酸消耗谱, 代谢组学, 高效液相色谱, 丝裂霉素, 紫杉醇

Abstract: An approach for quantitative determination of amino-acid consumption profiling in culture media by high performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed and validated, using o-phthalic dicarboxaldehyde (OPA)as the derivatizing reagent and norvaline as the internal standard. Mobile phase A was 10 mmol/L Na2HPO4- Na2B4O7 buffer (pH 7.95), and mobile phase B was acetonitrile-methanol-water (45:45:10, v/v/v). The linear elution program was 5%B at the start and 52%B at the end in 35 min. The 17 free amino-acids （FAAs） were separated satisfactorily in 33 min. Following HeLa cells incubation in conditioned medias of taxol (4 μmol/L) and mitomycin (75 μmol/L), respectively, with control for 24 h, the media 17 amino-acid consumption profilings were determined, and then analyzed by multivariate statistical analysis based on Matlab7.1 software platform. Relation analysis performed by partial least squares-discriminant analysis (PLS-DA) indicated that in comparison with the control group, the media amino-acid consumption profiling can distinguish the two anticancer drugs with different mechanisms, which provides a new perspective for the pre-classification of drug action mechanisms during the screening of new anticancer drugs. Meanwhile, the idea from the outer into the inner has convenient and economic characteristics.

Key words: amino-acid consumption profiling, HeLa cell, metabolomics, mitomycin, taxol, high performance liquid chromatography (HPLC)