摘要
“肾阳虚”为中医的基本证型之一,常伴随在不同疾病发生和发展过程中。机体系统的代谢物情况能真实反映机体在不同生理和病理状态下代谢终点的信息,并且随着分析仪器和数学统计方法的发展,代谢组学已经被广泛应用于疾病诊断、药物毒性评价等研究中。基于“肾阳虚”证的普遍性,我们假设“肾阳虚”证可能存在机体某种特定代谢轮廓的变化,利用代谢组学的方法能够捕捉到“肾阳虚”证所具有共性的代谢特征,并且参照中医辨证的策略,对未来临床疾病的分型、分期以及个性化治疗提供一个先进的分析方法。
本论文采用两种化学试剂诱导造模的方法建立“肾阳虚”证大鼠模型,对不同的时间点大鼠的尿样进行气相质谱联用仪(GC-MS)分析,同时对伴有“肾阳虚”证的两种典性疾病——慢性肾衰和慢性心衰患者的尿液进行GC-MS分析,并分别对“肾阳虚”大鼠和患者尿液的GC-MS原始图谱处理后进行多维和单维统计分析,发现与“肾阳虚”证发生和发展相关的代谢物谱的变化,随后,将“肾阳虚”证的动物模型和“肾阳虚”患者的代谢物变化信息进行比较,找到与“肾阳虚”证相关的代谢物变化规律。本论文的主要内容如下:
1、“肾阳虚”模型大鼠尿液的代谢组学研究
1.1采用三甲基硅烷(TMS)衍生化,GC-MS分析结合主成分分析(PCA)的方法,发现氢化可的松诱导的“肾阳虚”证大鼠在不同时间点,分别聚类在不同区域,通过结合T-test单维统计方法发现造模后10天,模型组与对照组比较,在尿液中有13个物质的相对浓度发生了显著升高,分别为酪胺、多巴胺、十四烷酸、半乳糖醛酸、十六烷酸、尿苷、3-羟脯氨酸、去甲肾上腺素、丙氨酸、古洛糖酸、胆固醇、丁酸、酪氨酸。主要涉及能量代谢、脂肪酸代谢和儿茶酚胺生物合成的变化。
1.2采用氯甲酸乙酯(ECF)衍生化GC-MS的分析方法测定腺嘌呤造模前后大鼠的尿样,得到尿中小分子代谢物的指纹图谱,结合模式识别(PCA和PLS-DA)等多维统计分析方法计算,峰响应信号的主成分分析图上显示腺嘌呤造模前后大鼠尿液的代谢谱发生了明显的变化。采用多维与单维统计结合的方法,找到与大鼠腺嘌呤造模前后分组相关的物质,在“肾阳虚”模型组尿液中代谢物浓度显著下降的是琥珀酸和油酸;显著升高的为酪氨酸、色氨酸、丙氨酸、甘氨酸、丝氨酸和苏氨酸。涉及到能量代谢、脂肪酸代谢、儿茶酚胺和色氨酸代谢变化。
本研究发现,在两种“肾阳虚”动物模型研究得到的差异性代谢物中,丙氨酸和酪氨酸的相对含量均表现为显著升高。
2、“肾阳虚”证患者尿液的代谢组学研究
2.1采用ECF衍生化,GC-MS结合多维和单维统计的方法,研究慢性心衰“肾阳虚”与慢性心衰非“肾阳虚”以及健康志愿者尿液之间代谢物谱的变化。在峰响应信号主成分分析图上显示慢性心衰“肾阳虚”、慢性心衰非“肾阳虚”以及健康志愿者之间呈现显著分离,分别找到与慢性心衰“肾阳虚”和慢性心衰非“肾阳虚”以及健康志愿者之间分组相关的差异物质。结果发现:与健康对照组比较,慢性心衰“肾阳虚”患者尿液中,琥珀酸、苹果酸、亚油酸和油酸浓度显著降低;缬氨酸、酪氨酸浓度显著升高,主要涉及到三羧酸循环、儿茶酚胺生物合成和脂肪酸代谢代谢通路的变化;慢性心衰“肾阳虚”和慢性心衰非“肾阳虚”比较的结果显示:被鉴定有显著性变化的代谢物的浓度在慢性心衰“肾阳虚”患者尿液中琥珀酸、苹果酸、柠檬酸、十六烷酸、花生四烯酸和油酸的相对含量显著下降,主要涉及三羧酸循环和脂肪酸代谢通路的变化,儿茶酚胺生物合成通路上的主要代谢物酪氨酸的浓度显著升高。?
2.2采用与研究慢性心衰“肾阳虚”相同的方法,进行了慢性肾衰“肾阳虚”、慢性肾衰非“肾阳虚”以及健康志愿者之间的代谢物变化。在峰响应信号的主成分分析图上显示慢性肾衰“肾阳虚”与健康组能显著的分开,慢性肾衰“肾阳虚”和健康对照组比较,柠檬酸、十六烷酸、油酸和亚油酸浓度显著降低,而丙氨酸和酪氨酸的浓度显著升高,涉及三羧酸循环、儿茶酚胺生物合成和脂肪酸代谢通路。?
两种“肾阳虚”证患者的尿液中油酸(下降)、酪氨酸(升高)的代谢物变化规律是一致的。
将“肾阳虚”动物模型和临床样本获得的尿液代谢组学结果进行比较,发现其共同特点是酪氨酸含量显著升高。同时,还发现慢性心衰“肾阳虚”、慢性肾衰“肾阳虚”和腺嘌呤诱导模拟的“肾阳虚”大鼠的显著变化代谢物中有2个变化规律完全相同,即油酸(下降)、酪氨酸(升高)。从本研究的结果可以初步推断,“肾阳虚”证是一种以酪氨酸(升高)代谢紊乱为主,还可能涉及其他代谢通路变化的一种特定代谢轮廓状态。该研究结果初步显示了代谢组学的方法对于具有共同代谢表型的疾病分型有很大潜力。
“kidney yang deficicency”is the one of the basic morbid situation in Chinese medicine, which usually existing in the onset and progression of many different diseases, such as chronic heart failure and renal failure. Metabolites can reflect the actual information of physiological and pathological status. With the development of modern chemical instrumental analysis and chemometrics analysis, metabonomics has been applied in lots of fields including morbid/disease diagnosis, toxicologic evaluation and so on. Therefore, we presume there might be specific changes of metabolic profiling in“kidney yang deficicency”and metabonomics method can find the changes.
In the article, we took different kinds of urine samples from patients and animals as the subjects to explain the variations of metabolites with“kidney yang deficicency”in the version of“metabonomics”.
We copyed the“kidney yang deficicency”animal model using ?hydrocortisone and adenine and urine samples of defferent timepoints were obtained. Subsequently, derivatized samples were infused in GC-MS in conjunction with modern multivariate statistical techniques to identify significant biochemical changes in different kinds of samples. The metabonomics approach enables us to visualize significant alterations in metabolite expression patterns as a result of hydrocortisone and adenine induced“kidney yang deficicency”rats metabolic responses. Alanine and tyrosine showed the same various trend in two kinds chemical regent induced“kidney yang deficicency”rats.
Clinical urine samples of“kidney yang deficicency”of chronic heart failure and“kidney yang deficicency”of renal failure were also used to process metabonomics study. The results showed that the concentration of olic acid and tyrosine had significiant change in the urine of“kidney yang deficicency”patients.
As a results, tyrosine concentration has significantly increased in both“kidney yang deficicency”rats and patients. The concentration of olic acid and tyrosine has the same various trend in adenine induced“kidney yang deficicency”rats and“kidney yang deficicency”patients of chronic heart failure and renal failure. Therefore, we may primarily concluded that the change of tyrosine concentration is a potential biomarker of“kidney yang deficicency”syndrom.
引文
1.沈自尹,中医药的一大特色-调整整体改善局部.中国中西医结合杂志,2006. 26(12): 1122-1125
2.于赫,郭丽新,肾阳虚证的生物化学和分子生物学研究进展.中医药信息,2005.22(6): 41-44
3.陈栋炜,蒙一纯,方湘娟等,肾阳虚证本质的现代医学研究进展和展望.现代中西医结合杂志,2005.14(23): 3175-3177
4. Basedovsky Ho, Sorkin E. Network of immune-neuroendocrine interactions. Chin Exp Immunl, 1977. 27 (1): 1- 12
5.沈自尹,有关证与神经内分泌免疫网络的研究.中医药学刊,2003. 21(1):10
6.宋春风,尹桂山,孙素菊,等.右归饮对大鼠下丘脑-垂体-肾上腺轴钙调素mRNA表达的影响.中国中医基础医学杂志, 2001.7 ( 3 ): 20-22
7.宋春风,尹桂山,赵建宏,等.补肾益气中药对肾阳虚大鼠下丘脑-垂体-肾上腺CaMPK II的影响.中国中医基础医学杂志, 2001.7 ( 8 ): 45-46
8.蔡定芳,沈自尹,张玲娟,等.右归饮对大鼠下丘脑-垂体-肾上腺-胸腺轴抑制模型的影响.中国免疫学杂志, 1994.10 (4): 236-239
9.沈自尹,黄建华,陈瑜,等.老年大鼠下丘脑-垂体-肾上腺-胸腺轴基因表达谱的研究.中国老年学杂志, 2004.24(2):125-127
10.章建民,沈翔,张红,等.“肾阳虚”小鼠免疫功能变化.浙江中医杂志, 2000. ( 6 ): 260
11.吕爱萍,李德新,崔家鹏,等.脾肾相关的分子生物学基础[.辽宁中医杂志, 2001.28 (3): 189-191
12.沈自尹,补肾法调节肾阳虚证T细胞凋亡的规律-重塑基因平衡.中西医结合学报, 2005. 5(2):
321-322
13.徐晓阳.运动性低血睾酮与肾阳虚大鼠能量代谢的比较研究.体育学刊,2000. ( 5 ): 41-44
14.卢德赵,沃兴德,施孟如等,激素型肾阳虚动物肝线粒体蛋白质组与能量代谢相关性.中国生物化学与分子生物学报,2005. 21 (6) : 807-814
15.沈自尹,张新民,林伟等,基于基因表达谱数据建立肾虚证量化数学模型.中国中西医结合杂志, 2008. 28(2): 131-134
16. Nemeth E F. Ca2 + receptor-dependent regulation of cellular functions . Int union Physiol Sci, 1995. 10 : 126
17.宋春风,郑师陵,吕佩源等,补肾中药对肾阳虚大鼠下丘脑-垂体-肾上腺轴、血淋巴细胞Ca2 +和血清钙的影响.中国中医基础医学杂志,2002. 8(5): 34-36
18. Wi11iams, J. E. et al., Guidelinesfortheevaluation and management of heart failure·Report of the Ameriean College of Cardiology/Ameriean Heart Association Task Foreeon Praetice Guidelines (Committee on Eva1uationand Management of HeartFai1ure).Am CollCardiol, 1995. 26(5): 137-139
19. Gheorghiade, M. et al., Chronic heart failure in the united States :a manifestation of coronary artery disease.Cireulation,1998.97(3): 282-289
20. Hok, K. L. et al., Thee Pidemiology of heart failure:the Framingham study.Am CollCardiol, 1993. 22: 6-13
21.顾东风,黄广勇,何江,等,中国心力衰竭流行病学调查及其患病率.中华心血管病志,2003. 31(1): 3-6
22.曹贵民,中医对心力衰竭的认识与治疗.陕西中医学院学报,1997,20(3): 6-10
23.程丑夫,张炜宁,心衰的中医病证归属浅析.山东中医药大学学报, 1998.22(2): 107-108
24.吴勉华,充血性心力衰竭中医病机探讨.南京中医药大学学报自然科学版,2001.17(4): 206- 209
25. Baig, M. K. et al., The pathophysiology of advanced heart failure. Am Heart , 1998.135: 216
26. Bing, O.H.. Hypothesis apoptosis machanism for the transition to heart failure with chronic pressure overload. J Mol Cell Caediol, 1994. 26: 943
27.Eohn J N. Structural changes in cardiovaseular disease. Am J cardioz,1995: 76:34
28. Weber, K. J. et al., Structural remodeling of the heart by fibrous tissue: role of eireulating hormones and loeally produced peptides.Eur Heart, 1995.16(1): 12
29. Jdaly, P. A. et al., Eholamine, andtloe pathophysiology of heart failure.Circulation, 1990.82(suppl): 135
30. Urata, H. et al. Cellular loealization and regional distribution of anangiotens in for minge hynase in the heart. Clin Invest,1993. 91: 1269
31. Aukrkst, P. et al., Cytokinc network in congestive heart failure secondary toisehemieoridio pathiedilated cardiomyopathy.AmCardiol, 1999.83: 376-382
32. FoleyRN, et al. epidemiology of cardiovaseular disease in chroni renal disease. Am J Kidney Dis, 1998.32[SuPpl3]: 112-119
33. BostomA, et al., Hyperhomoeyste in emia in end stage renal disease: Prevalenee,etiology,and Potential relationship to artherosclerotieouteomes. Kidney Int1997: 52:10
34.中华医学会肾脏病分会透析移植登记工作组. 1999年度全国透析移植登记报告.中华肾脏病杂志,2001.17: 77-78
35.Ma KW, et al.,Cardiovascular risk factors in chronic renal failure andhemodialysis populations. Am J KidDis, 1992.19: 505-513
36. Nicholson, J.K., et al., Metabonomics: a platform for studying drug toxicity and gene function. Nat Rev Drug Discov, 2002. 1(2): 153-161
37. Minjun Chen, et al., Metabonomic Study on the Biochemical Profiles of A Hydrocortisone-Induced Animal Model. J Proteome Res, 2005. 4(6): 2391-239638. Lindon JC, et al.,Contemporary issues in toxicology the role of metabonomics in toxicology and its evaluation by the COMET project Toxicol Appl Pharm.2003,187: 137-145
39. Nicholson, J.K., et al. , 'Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica, 1999. 29(11): 1181-1189
40. Fiehn, O., Metabolomics--the link between genotypes and phenotypes. Plant Mol Biol, 2002. 48(1-2): 155-171
41. Weckwerth, W., Metabolomics in systems biology. Annu Rev Plant Biol, 2003. 54: 669-89
10. Fiehn, O., et al., Metabolite profiling for plant functional genomics. Nat Biotechnol, 2000. 18(11): 1157-61
42. Raamsdonk LM, et al., A functional genomics strategy that uses matabolome data to reveal the phenotype of silent mutations.Nat Biotechnol, 2001.19(1): 45-50
43. Trethewey RN.Gene discovery via metabolic profiling.Curr Opin Biotechnol. 2001.12:135-138
44. Reo, N.V., NMR-based metabolomics. Drug Chem Toxicol, 2002. 25(4): 375-82
45. Holmes E., et al., The application of NMR-based metabonomics in neurological disorders. NeuroRx, 2006. 3(3): 358-72
46. Plumb R.S., et al., Metabonomics: the use of electrospray mass spectrometry coupled to reversed-phase liquid chromatography shows potential for the screening of rat urine in drug development. Rapid Commun Mass Spectrom, 2002. 16(20): 1991-6
47. Rohrich J. et al., Determination of amphetamine and methylenedioxy-amphetamine-derivatives in hair. Forensic Sci Int, 1997. 84(1-3): 179-88
48. Lee H.B., et al., Determination of endocrine-disrupting phenols, acidic pharmaceuticals, and personal-care products in sewage by solid-phase extraction and gas chromatography-mass spectrometry. J Chromatogr A, 2005. 1094(1-2): 122-9
49. Pons A., et al., Sequential GC-MS analysis of sialic acids, monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives. Biochemistry, 2003. 42(27): 8342-53
50. Husek P., Chloroformates in gas chromatography as general purpose derivatizing agents. J Chromatogr B Biomed Sci Appl, 1998. 717(1-2): 57-91
51. Frison G., et al., Gas chromatography/mass spectrometry determination of amphetamine-relateddrugs and ephedrines in plasma, urine and hair samples after derivatization with 2,2,2-trichloroethyl chloroformate. Rapid Commun Mass Spectrom, 2005. 19(7): 919-927
52. Berger u., et al, Two trace analytical methods for determination of hydroxylated PCBs and other halogenated phenolic compounds in eggs from Norwegian birds of prey. Anal Chem, 2004. 76(2): 441-452
53. Tweeddale H, et al., Effect of slow growth on metabolism of Escherichia colias as revealed by global metabolite pool(“Metabolome”) analysis.J bacteriology, 1998. 180: 5109-5116
54. Aharoni A, et al., Non targeted metabolic profiling using fourier transformation cyclotron mass spectrometry(FTMS). Omics: A Journal of Intigrative biology, 2002.6(3): 217-234
55. Zaikin V.G. et al., Review: derivatization in mass spectrometry-6. Formation of mixed derivatives of polyfunctional compounds. Eur J Mass Spectrom (Chichester, Eng), 2005. 11(6): 611-36
56. Halket J.M. et al., Derivatization in mass spectrometry--1. Silylation. Eur J Mass Spectrom , 2003. 9(1): 1-21
57. Lefevere, M.F., et al., Metabolic profiling of urinary organic acids by single and multicolumn capillary gas chromatography. J Chromatogr Sci, 1989. 27(1): 23-29
58. Fraser, A.D., et al., urinary screening for midazolam and its major metabolites with the Abbott ADx and TDx analyzers and the EMIT d.a.u. benzodiazepine assay with confirmation by GC-MS. J Anal Toxicol, 1991. 15(1): 8-12
59. Matsunaga, S. et al., Determination of alpha- and beta-hydroxycarbonyls and dicarbonyls in snow and rain samples by GC/FID and GC-MS employing benzyl hydroxyl oxime derivatization. Anal Chem, 2000. 72(19): 4742-4746
60. Smith, C.A., et al., XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem, 2006. 78(3): 779-87
61. Idborg, H., et al., Metabolic fingerprinting of rat urine by LC/MS Part 2. Data pretreatment methods for handling of complex data. J Chromatogr B Analyt Technol Biomed Life Sci, 2005. 828(1-2): 14-20
62. Jonsson, P., et al., Predictive metabolite profiling applying hierarchical multivariate curve resolution to GC-MS data--a potential tool for multi-parametric diagnosis. J Proteome Res, 2006. 5(6): 1407-14
63. Yang, J., et al., [Metabonomics and its applications]. Sheng Wu Gong Cheng Xue Bao, 2005.21(1): 1-5
64. Ebbels, T.M., et al., Prediction and classification of drug toxicity using probabilistic modeling of temporal metabolic data: the consortium on metabonomic toxicology screening approach. J Proteome Res, 2007. 6(11): 4407-22
65. Chen, M., et al., Metabonomic study of aristolochic acid-induced nephrotoxicity in rats. J Proteome Res, 2006. 5(4): 995-1002
66. Mitchell, S., E. et al., Metabonomics and medicine: the Biochemical Oracle. Biologist (London), 2002. 49(5): 217-21
67. Dieterle, F., et al., Application of metabonomics in a compound ranking study in early drug development revealing drug-induced excretion of choline into urine. Chem Res Toxicol, 2006. 19(9): 1175-81
68. Yang, J., et al., Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases. J Chromatogr B Analyt Technol Biomed Life Sci, 2004. 813(1-2): 59-65
69.毛煜,袁伯俊,代谢组学的研究现状与展望.中国新药杂志,2007. 16(13): 1005-1010
70.陈慧梅,刘志红,代谢组学及其研究方法和应用.肾脏病与透析肾移植杂志,2005.14(1): 59-64
71.杨军,宋硕林,Jose Castro-Perez等,代谢组学及其应用.生物工程学报,2005.21(1): 1-5
72.施钰琳,王小琴,代谢组学及其应用研究新进展.医学综述,2007.13(20): 1543-1545
73.许国旺,路鑫,杨胜利,代谢组学研究进展.中国医学科学院学报,2007.29 (6) : 701 - 711
74.徐昊,林东海,刘昌孝,代谢组学研究现状与展望.药学学报,2005.40 (9) : 769 - 774
75.董继扬,徐乐,曹红婷等,代谢组学数据分析方法及在糖尿病研究中的应用.波谱学杂志, 2007.24(4): 3811-3813
76.李晶,吴晓健,刘昌孝等,代谢组学研究中数据处理新方法的应用.药学学报,2006. 41 (1) : 47 - 53
77.林艳萍,司端运,刘昌孝,液相色谱和质谱联用技术结合化学计量学应用于代谢组学的研究进展.分析化学,2007.35(10): 1535-1540
78.薛骞,代谢组学技术:中医药发展的重大契机.中华中医药学刊,2008. 26(1): 176-177
79.罗和古,陈家旭,代谢组学技术与中医证候的研究.中国中医药信息杂志,2007.14(5): 3-5
80.谢毅强,牛乾,代谢组学与中医基础理论的研究进展.医学综述,2008.14(4): 533-535
81.贾伟,蒋健,刘平等,代谢组学在中医药复杂理论体系研究中的应用.中国中药杂志,2006.31(8): 621-623
82.黄建华,沈自尹,吴斌等,从基因表达谱和代谢组学角度探讨淫羊蕾总黄酮延缓衰老的效应及机制.中国中西医结合杂志,2008. 28(1):47-50
83.梁茂新,《中医证研究的困惑与对策》,北京,人民出版社. 2000.126
84.张天奉,张建军,吕金阳,走出中医证候客观化研究的误区,中国中医基础医学杂志,2004. 10(11):21-22
1.沈自尹,肾的研究(续集).上海:上海科技出版社,1990: 9-17
2.吴启端,熊带水,梁文能,肾阳虚动物模型的研究概况.中国实验方剂学杂志,2001. 7(6): 54-56.
3.周文江,姚菊芳,彭秀华,肾阳虚证大鼠模型的建立.实验动物与比较医学,2007. 27(4): 242-243
4.杨裕华,李震,肾阳虚动物模型及其诊断指标研究近况.辽宁中医药大学学报,2007. 9(5): 40-42
5.宋春风,尹桂山,吕佩源,肾阳虚证的中西医结合研究概况.中国中医基础医学杂志,2001. 7(7): P. 76-79
6.李广曦,肾阳虚证动物模型的造模方法及其相关指标回顾.中国中医基础医学杂志,2000. 6(4): 46-54
7.陈英华,欧阳轶强,孙琪等,肾阳虚证动物模型规范化研究中诊断指标选择的初步探讨.中国中医基础医学杂志,2003. 9(10): 26-30
8.陈英华,欧阳轶强,孙琪等,肾阳虚证动物模型造膜方法综述.中国医药学报,2003. 18(6): 370-372
9.张伟荣,肾阳虚证动物实验研究简述及展望.中医药学刊,2006. 24(10): 1809-1811
10.邝安堃,吴裕,丁霆,等某些助阳药对于大剂量皮质素所致耗竭现象的影响.中华内科杂志,1963. (2) :113
1.沈自尹,陈响中,陈剑秋等,“肾阳虚”证的下丘脑-垂体-甲状腺轴的初步研究.上海中医药杂志, 1982.2: 43-45
2.徐建华,论肾阴虚证、肾阳虚证等研究的突破口.中国医药学报, 2002.17(2): 86-88
3.王建红,伍庆华,刘海云等,肾阳虚大鼠垂体-甲状腺轴功能动态分析.辽宁中医杂志, 2006. (33) 6: 751-752
4.伍庆华,王建红,刘海云等,肾阳虚大鼠垂体-甲状腺轴与垂体-性腺轴相关性研究.江苏中医药, 2007. (39) 3: 56-57
5.伍庆华,王建红,祁风义等,肾阳虚与下丘脑-垂体-靶腺轴研究概述.江西中医学院学报, 2006.(18)6: 73-74
6.董兴刚,张庆怡,陈以平,肾阳虚证的研究进展.中国中西医结合肾病杂志,2003.(4)5: 299-300
7.沈自尹,衰老-生理性肾虚证的HPAT轴分子网络调控研究.中国中西医结合杂志, 2004. 24(9): 841-843
8. Tischler, A. S., Perlman, R. L.,Morse, G. M., Sheard, B. E, J. Neurochem. 1983. 40 (2):364-370
9. Mckee, T., McKee, J. R. Biochemistry: An introduction; MacGraw-Hill Co., Inc.: Sao Paulo, 2001, p 207.
10. Otten, U., Thoenen, H. Mol. Pharmacol. 1976. 12 (3):353-361.
11. Tischler, A. S., Perlman, R. L., Morse, G. M. J. Neurochem. 1983.40 (2): 364-370.
12. Minjun Chen, Liping Zhao, Wei Jia. Metabonomic Study on the Biochemical Profiles of A Hydrocortisone-Induced Animal Model. Journal of Proteome Research . 2005. 4: 2391-2396.
1.邝安堃,吴裕,丁霆等.某些助阳药对于大剂量皮质素诱导耗蝎现象的影响.中华内科杂志,1963. (2) :651
2.郑平东,朱燕俐,丁名城等,腺嘌呤诱发“肾阳虚”动物模型的研究.中国医药学报,1990. 5(3): 68-73
3.周文江,姚菊芳,彭秀华,肾阳虚证大鼠模型的建立.实验动物与比较医学,2007. 27(4): 242-243
4.宋春风,尹桂山,吕佩源,肾阳虚证的中西医结合研究概况.中国中医基础医学杂志,2001. 7(7): P. 76-79
5.郑平东,朱燕俐,丁名城等,腺嘌呤诱发睾丸功能损害肾阳虚模型的研究.中国医药学报,1989. 4(3): 67-69
6.沈自尹,中医虚证辨证参考标准.中西医结合杂志,1986.(10) :598
7.傅晓晴,武一曼,陈振彬等,腺嘌呤制作肾阳虚型慢性肾功能衰竭大鼠模型的电镜病理学研究.福建中医学院学报, 2002. 12(3): 41-43
8.傅晓晴,林若勤,武一曼等,腺嘌呤制作肾阳虚型慢性肾功能衰竭大鼠模型的生化研究.福建中医学院学报,2003 .13(1): 22-24
9.吕爱平,李德新,易杰等,脾肾阳虚模型大鼠脂质过氧化与抗氧化相关性的比较研究.中国中医基础医学杂志,2006.6(12):785-786
10.曾庆成,交泰丸治疗心肾不交病症浅识.湖北中医杂志,1999 . 21 (7) : 325
11.吕爱平,李德新,崔家鹏等,脾肾相关的分子生物学基础.辽宁中医杂志,2001. 28(3): 189-191
1. Qiu, Y., et al., Application of ethyl chloroformate derivatization for gas chromatography-mass spectrometry based metabonomic profiling. Anal Chim Acta, 2007. 583(2): 277-83
2. Husek, P.Chloroformates in gas chromatography as general purpose derivatizing agents. J Chromatogr B, 1998.717:57-91
3. Brindle, JT, et al., Application of chemometrics to H-1NMR spectroscopic data to investigate a relationship betweenhuman serum metabolic profiles and hypertension.Analyst. J Chromatogr B, 2003.128: 32-36
4. Dumas, ME, et al., Assessment of analytical reproducibility of 1H NMR spectroscopy based metabonomics for large-scale epidemiological research: the INTERMAP Study.Anal Chem., 2006,78: 2199-2208
5. Espina, JR, et al., Detection of in vivo biomarkers of phospholipidosis using NMR-based metabonomic approaches. Magnet Reson Chem., 2001. 39: 559-565
6. Fiehn, O, et al., Identification of uncommon Plant Metabolites Based on Calculation of Elemental Compositions using Gas Chromatography and Quadrupole Mass Spectrometry.Anal Chem., 2000. 72: 3573-3580
7.沈自尹,补肾法调节肾阳虚证T细胞凋亡的规律-重塑基因平衡.中西医结合学报, 2004.2(5): 321-322
8.沈自尹,张新民,林伟等,基于基因表达谱数据建立肾虚证量化数学模型.中国中西医结合杂志,2008.28(2): 131-134
9.沈自尹,肾阳虚证的定位研究.中国中西医结合杂志,1997.17(1): 50-52
10. Husek P., Amino acid derivatization and analysis in five minutes. FEBS Lett.1991, 280:354-356.
11. Husek P.Simultaneous profile analysis of plasma amino and organic acids by capillary gas chromatography.J Chromatogr B.1995,669: 352-357
12.邵命海,肖静,王毅兴等,从“肾主生殖”角度评价腺嘌呤与氢化可的松诱导的肾阳虚模型.上海中医药杂志,2008.42(2):57-59
13.郑平东,朱燕俐,丁名城等,腺嘌呤诱发“肾阳虚”动物模型的研制.中国医药学报, 1990.5(3):68-73
14.卢德赵,沃兴德,施孟如等,激素型肾阳虚动物肝线粒体蛋白质组与能量代谢相关性.中国生物化学与分子生物学报,2005. 21 (6) : 807-814
15.沈自尹,黄建华,陈伟华,以药测证对肾虚和肾阳虚大鼠基因表达谱的比较研究.中国中西医结合杂志,2007. 27 (2): 135-137
16.傅晓晴,林若勤,武一曼等,腺嘌呤制作肾阳虚型慢性肾功能衰竭大鼠模型的生化研究.福建中医学院学报,2003 .13(1): 22-24
17.刘尚全,肾阳虚证临床及实验研究回顾.生命科学趋势,2003. 1(4):92-101
1.徐燕,阮小芬,金涛等,慢性心力衰竭患者心肾阳虚本质初探.上海中医药大学学报,2007.21(4): 45-46
2.杨卫彬,王金彭,慢性肾衰合并心衰的危险因素及中医治疗思路探讨.中国中医药信息杂志,2002. 9 (5): 52-53
3. Cleland JC,et al. Has the survival of the heart failure population changed?Lessons fromtrials.Am J Cardiol,1999.83: 112-119
4. KlahrS,etal.The role of vasoactive compounds growth factors and cytokines in the Porgression of renal disease.Kidney Int,2000. 75: 1-7
5. Terzi F,, et al. using transgenie mice to analyze the mechanisms of prorgerssion of chronic renal failure. J Am Soe Nephrol, 2000.11: 144-148
6.刘宏伟.慢性肾衰中医辨证分型和疗效判断标准.中医药信息,1991.8: 27-28
7.杨宏,江骥,胡蓓.慢性肾衰时蛋白质和氨基酸代谢的研究.中国临床营养杂志,2001.9(3): 175-177
8. Lewen Jia, Jing Chen, Peiyuan Yin, et al. Serum metabonomics study of chronic renal failure by ultra performance liquid chromatography coupled with Q-TOFmass spectrometry. Metabolomics, 2008. 4: 183–189
9. Xiumei Tao,Yumin Liu, Yihuang Wang, et al. GC-MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia. Ananlytical and bioanalytical chemistry. DOI: 10.1007/s00216-008-2220-8
10.杨卫彬,王金彭,慢性肾衰合并心衰的危险因素及中医治疗思路探讨.中国中医药信息杂志,2002. 9 (5): 52-53
11.杨云松,王文刚,近十年来慢性肾衰的中医、中西结合研究状况及思路概述.中医药学报,2007.35(6): 29-33
12.谢桂权,慢性肾衰中医治疗整体观初探.世界中医药,2007. 2 (1): 10-12
13.刘旭生,刘兴烈,刘敏雯等,慢性肾衰中医证型分布特征的临床研究.陕西中医,2007.28(8): 964-969
14.李慧,慢性肾衰的中医分型论治.浙江中医学院学报,2005.29(6): 8
15.薛青,王小娟,慢性肾衰的中医临床研究进展.现代中西医结合杂志,2002. 11(2): 129-131
16.陈哲林、许金诚,心力衰竭患者血清肿瘤坏死因子、白细胞介素6和白细胞介素-6受体水平的测定及临床意义.广东医学院学报, 2002.20(4): 276-277
17.罗文浩,丁宇栋,鲍逸民.慢性充血性心力衰竭血清甲状腺素变化的意义.苏州大学学报(医学版),2005. 25 (6):1101-1102
18. Hillege HL, et al. Renal function,neurohumoral activation,and survival in patients with chronic heart failure.Circulation,2000.102: 203- 210
19.沈自尹,肾阳虚证的定位研究.中国中西医结合杂志,1997.17(1): 50-52
20.郭传明,杨丽霞,王燕等,慢性心衰患者胰岛素抵抗与血清瘦素的相关性研究.西南国防医药,2007. 17(4): 428-429
21. Swanj W, Walton C,Godsland I F ,et al. Insulin resistance in chronic heart failure. Eur Heart J , 1994. 15(12) : 1528-1530
22.曾洪,谭焕源,蔡新.左旋卡尼汀治疗尿毒症慢性心衰的临床观察.广西医学,2005. 27(11): 1821-1822
23. Terzi F,, et al. using transgenie mice to analyze the mechanisms of prorgerssion of chronic renal failure. J Am Soe Nephrol,2000.11: 144-148
24.马济佩,何立群,郑平东,慢性肾衰患者尿毒症毒素水平与中医辨证分型之间关系的临床研究.四川中医,2005.23(2): 21-22
25.邓声京.脂质代谢异常与慢性肾小球损伤.国外医学·泌尿系统分册,2005.25: 99-103
26.袁成凌,姚建铭,余增亮.花生四烯酸及其代谢物的生物学作用.中国药物化学杂志, 2000.10(1):75-78
27.杨宏,江骥,胡蓓.慢性肾衰时蛋白质和氨基酸代谢的研究.中国临床营养杂志,2001.9(3): 175-177
28. Yonova D. Serum homocysteine in patients with renal disease and chronic renal failure on conservative and dialysis treatment. Nephrology, Dialysis and Transplantation, 2004.10 (1): 34-36
29. Cano N. et al. Effect of chronic renal failure with metabolic acidosis on alanine metabolism in isolated liver cells. Clinical Nutrition,2004. 23 (3): 317-324
30. Pawlak. D. et al. Tryptophan metabolism via the kynurenine pathway in experimental chronic renal failure . Nephron , 2002. 90 (3): 328-335
1.林景超,李后开,周明眉等,代谢组学、药物代谢组学与中医药现代化.中国医学科学院学报,2007. 29 (6): P.818– 822
2.李后开,王一煌,贾伟等,药物基因组学、药物代谢组学与辨证施治.世界科学技术—中医药现代化—创新论坛,2007.9(2):120—126
3.徐燕,阮小芬,金涛等,慢性心力衰竭患者心肾阳虚本质初探.上海中医药大学学报,2007.21(4): 45-46
4.李娟,王淑秋,杨洪宇等,心衰患者TNF-α、生长激素与下丘脑-垂体-甲状腺轴的相关性,心血管康复医学杂志,2006. 14(1):17- 19
5.杨卫彬,王金彭,慢性肾衰合并心衰的危险因素及中医治疗思路探讨.中国中医药信息杂志,2002. 9 (5): 52-53
6.章建民,张红,周羽琪等,肾阳虚模型小鼠红细胞膜磷脂脂肪酸含量变化观察.浙江中医杂志,2000.3:128-129
7.黄建华,沈自尹,吴斌等,从基因表达谱和代谢组学角度探讨淫羊蕾总黄酮延缓衰老的效应及机制.中国中医细节和杂志,2008. 28(1): 47-50
8.谢桂权,慢性肾衰中医治疗整体观初探.世界中医药,2007. 2 (1): 10-12