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大鼠肝郁脾虚证的代谢组学研究
引用本文:罗和古,丁杰,岳广欣,陈家旭.大鼠肝郁脾虚证的代谢组学研究[J].中西医结合学报,2007,5(3):307-313.
作者姓名:罗和古  丁杰  岳广欣  陈家旭
作者单位:北京中医药大学中医诊断系,北京,100029
摘    要:目的:研究慢性束缚应激大鼠(肝郁脾虚模型)的血浆代谢表型改变,开展中医证候学的研究,试图通过对慢性束缚应激大鼠代谢物组的共性分析和生物标记物的发现,寻找肝郁脾虚证候的生物学本质,探讨代谢组学在中医证候学研究中的应用。方法:选用实验用二级雄性SD大鼠24只,随机分为4组:7 d正常对照组(A组)、21 d正常对照组(B组)、7 d模型组(C组)和21 d模型组(D组),每组6只。以慢性束缚方法制作应激大鼠模型。分别于第8天和第22天麻醉后心室取血,用Varian UnityInova 600 M超导傅立叶变化核磁共振(nuclear magnetic resonance,NMR)波谱仪检测。自由感应衰减信号经过32 k傅立叶变换转为一维NMR谱图。调用VNMR软件中的程序将1H谱按默认值,分别从4.5~0.5 ppm(弛豫时间编辑)以及6.0~0 ppm(扩散编辑),每段为0.04 ppm,进行分段积分,将积分数据归一化后,以文本文件或Excel文件贮存,用于模式识别分析。将上述得到的数据文件利用Si mca-P 10.0软件包进行主成分分析。结果:大鼠血浆1H NMR谱的主成分分析,各组动物代谢谱各不相同,与已知应激状态下动物体内代谢的调节过程及结果相一致。正常对照组与模型组相比较发现,醋酸、乳酸、酪氨酸、低密度脂蛋白和3.44 ppm的未知化合物的谱峰峰形改变较为明显。这些发生改变的代谢物可以作为肝郁脾虚证的生物标志物做进一步的研究。结论:各组大鼠血浆1H NMR代谢谱之间存在差异,而且可能从代谢组学分析中找出特异的标志性代谢产物,阐释中医证候的生物学本质。代谢组学分析是一种有良好发展前景的中医证候学研究方法。

关 键 词:代谢组学  模式识别  核磁共振  中医  大鼠
文章编号:1672-1977(2007)03-0307-07

Metabonomic study of syndrome of liver qi stagnation and spleen deficiency in rats
He-gu LUO,Jie DING,Guang-xin YUE,Jia-xu CHEN.Metabonomic study of syndrome of liver qi stagnation and spleen deficiency in rats[J].Journal of Chinese Integrative Medicine,2007,5(3):307-313.
Authors:He-gu LUO  Jie DING  Guang-xin YUE  Jia-xu CHEN
Institution:Department of Diagnostics of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
Abstract:Objective: To determine the changes of the plasma metabolic phenotype in rats with chronic restraint stress(rats with syndrome of liver qi stagnation and spleen deficiency),so as to reveal the biological features of the syndrome of liver qi stagnation and spleen deficiency,and to examine potential application of metabonomic analysis in studies of syndromes in traditional Chinese medicine.Methods: Twenty-four male Sprague-Dawley rats were randomly divided into four groups: group A,7 d normal control group;group B,21 d normal control group;group C,7 d stress group;and group D,21 d stress group,with 6 rats in each group.Chronic restraint was used to induce stress in rats.Blood was collected from the cardio-ventricle under anesthesia on the 8th(groups A and C) or 22nd day(groups B and D) and detected by using the Fourier variable superconducting nuclear magnetic resonance(NMR) spectrometer(Varian UnityInova 600 M).Free induction decay signals were transferred into one-dimensional NMR spectrogram via 32 k Fourier transformation.Segmental integral calculus(0.04 ppm per segment) was performed from 4.5-0.5 ppm(Carr-Purcell-Meiboom-Gill,CPMG) or 6.0-0 ppm(longitudinal eddy-delay,LED) as defaulted 1H spectra values by using the VNMR software.Data were saved as text or excel files after normalization and then used for pattern recognition analyses.All the data were analyzed by principal component analysis(PCA) using the SIMCA-P 10.0 software(Umetrics AB,Umea,Sweden).Results: The PCA analysis of rat plasma 1H NMR spectra revealed different metabolic spectra between stress and control groups,which were consistent with alterations of in vivo metabolisms in rats under stress stimuli.Compared with the normal control group,rats with repeated stress displayed significant changes in spectral peak shapes of acetate,lactate,tyrosine,low-density lipoprotein,and unknown compounds(3.44 ppm).These altered metabolites can be used as biomarkers of syndrome of liver qi stagnation and spleen deficiency for further studies.Conclusion: The 1H NMR spectra of metabolites in the rat blood are differentially changed after chronic stress.Specific,characteristic metabolic products can be identified by analyses of metabonomics,which lead to interpretation of biological feature of Chinese medicine syndromes.Therefore,metabonomic analysis is an approach with good development prospects to studies of TCM syndromes.
Keywords:metabonomics  pattern recognition  nuclear magnetic resonance  traditional Chinese medicine  rats
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