从表征和尿液代谢组学角度判别2型糖尿病大鼠中医证候属性的实验研究
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摘要
目的：建立并优化糖尿病大鼠表征采集表和表征采集方法；建立高脂饲料叠加链脲佐菌素诱发的2糖尿病大鼠模型,探索大鼠证候属性的判断方法；探索具有补脾益气、祛痰化瘀功效的中药复方干预病证结合2型糖尿病大鼠的疗效及其评价方法；从尿液代谢组学的角度阐释病证结合2型糖尿病大鼠的证候相关生物学基础和中药复方干预大鼠模型的药效机制。
方法：实验研究一即腹腔注射链脲佐菌素诱发糖尿病大鼠模型,初步建立病证结合糖尿病大鼠模型的表征采集表,检测阳性表征及血糖、激素、代谢笼、体温、体重等指标,以临床证候诊断标准及文献报道的相关专家观点为大鼠证候属性的判断依据,对糖尿病大鼠进行初步的证候属性判断；实验研究二即高脂饲料叠加链脲佐菌素复制2型糖尿病大鼠模型,根据动物实验一所建立的表征采集表对大鼠模型进行表征采集,检测血糖、血脂、血流变及激素等指标,结合上述结果判断大鼠模型的证候属性；实验研究三根据方证相应理论,采用具有补脾益气、祛痰化瘀功效的中药复方(高、中、低三个剂量组)干预2型糖尿病大鼠,检测血糖、血脂、胰腺和肝脏病理变化等一般理化指标,结合阳性表征结果,初步评价中药复方的疗效；实验研究四采用气质联用技术对2型糖尿病大鼠(中药高剂量组、西药组、模型组、对照组)的尿液进行检测,根据尿液代谢物的结果,阐释不同组、前后两个时间点(时间点一代表脾气亏虚证为主的阶段,时间点二代表痰湿瘀滞证为主的阶段)大鼠模型的证候及其演变规律的相关生物学基础,以及中药复方干预大鼠模型的疗效机制。
结果：实验研究一建立并优化了实验性糖尿病大鼠的表征采集表,制定了操作简便的表征采集方法；实验研究二以临床证候诊断标准及文献报道的相关专家观点为依据,以业已建立的采集表征表为主要工具,初步建立了病证结合2型糖尿病大鼠的证候属性判断方法。实验研究二建立的2型糖尿病大鼠模型,第1-3周模型组大鼠表征出现形体消瘦、行为呆滞、毛色黄、耳、鼻、唇、舌、爪颜色淡白和尾部重度角化,抓力下降及体质量减低,但饮食量、饮水量、尿量、体温与对照组相比没有差别。第4-6周模型组大鼠部分表征发生进一步变化,与前3周相比,上述阳性表征的比率有上升趋势,并且舌色暗红及大便黏滞松软比率明显增加；实验研究三建立的2型糖尿病大鼠模型经过中药复方干预6周后,一般理化指标和阳性表征均有不同程度的改善。中药高剂量组大鼠模型的血糖、胆固醇、甘油三酯、低密度脂蛋白、尿素氮、谷丙转氨酶和谷草转氨酶水平明显降低。在中药复方改善大鼠模型表征方面,中药高剂量组大鼠的行为活跃比率、耳鼻唇爪颜色淡白比率、舌暗红比率、以及大便黏滞松软比率均有明显改善,出现表征改善的时间点多在中药复方干预的中后期；实验研究四尿液代谢物鉴定结果显示,与时间点一比较,时间点二模型组大鼠尿液中二羟基苯甲酸、L-抗坏血酸、D-葡萄糖酸十八烷酸和戊二酸含量均明显增加；时间点二中药高剂量组大鼠的尿液中L-抗坏血酸含量减少,
结论：实验研究一制定的表征采集表可全面、动态地采集糖尿病大鼠的宏观表征,大鼠成模后第3-7周的表征基本稳定,结合一般理化指标,初步推测模型组大鼠证候特征为早期胃热炽盛证为主,后期脾气亏虚证为主。实验研究一大鼠的证候属性尚不属于文献报道的糖尿病临床常见证型范畴；实验研究二结合大鼠阳性表征和一般理化指标,发现2型糖尿病大鼠第1-6周的表征基本稳定,初步推测模型组大鼠证候特征为前期脾气亏虚证为主,后期痰湿瘀滞证为主。实验研究二大鼠的证候属性属于糖尿病临床常见证型范畴,可作为方证相应研究的合格载体；实验研究三显示高剂量中药复方具有改善2型糖尿病大鼠表征和一般理化指标的作用,从方证相应的角度证明了2型糖尿病大鼠的证候属性可能为脾气亏虚、痰湿瘀滞证。笔者认为从表征和一般理化指标两个方面对2型糖尿病大鼠进行证候疗效评价具有可行性；实验研究四显示,大鼠尿液代谢物的变化可在一定程度上揭示病证结合2型糖尿病大鼠的证候相关生物学基础及中药药效的机制。
Objectives:To establish the physical-sign-collecting form and its collection methods; To explore the method of estimating the syndrome of animal models combining disease with syndrome of Type 2 DM, which are duplicated with high-fat diet and Streptozotocin (STZ); To evaluate the curative effect of formulas on animal models combining disease with syndrome of Type 2 DM; To explore the essence of syndrome of animal models and the mechanism of curative effect of formulas by means of urine metabonomics.
Methods:The research on physical signs and syndrome of diabetic rat models built up with STZ was carried out, the physical-sign-collecting form and its collection method were established. Blood glucose, blood lipid, hormone, body temperature, body weight and so on were measured. The diagnosis of syndrome of rat models was made in the basis of combination of diagnostic standard of clinical syndrome and viewpoints of specialists; The research on physical signs and syndrome of diabetic rat models built up with high-fat diet and STZ was carried out. The physical signs were collected according to the former physical-sign-collecting form. Blood glucose, blood lipid, hormone and so on were measured. The diagnosis of syndrome of rat models was made in the same method; The research on evaluating the curative effect of formulas applied on rat models built up with high-fat diet and STZ was carried out. The estimation of curative effect of formulas was made by means of physical-sign-collection and measurement of blood glucose, blood lipid and pathological changes; The research on applying GC-MS to analyzing the urine of formula-treating rat models built up with high-fat diet and STZ was carried out. The differences of metabolites in different groups and time were analyzed and explained the biological basis of syndrome and mechanism of curative effect.
Results:The physical-sign-collecting form and its collection method were established through the research on physical signs and syndrome of diabetic rat models built up with STZ; On the basis of collecting physical signs and measuring blood, the method of diagnosing syndrome of rat models built up with high-fat diet and STZ had been established according to the combination of diagnostic standard of clinical syndrome and viewpoints of specialists. In the former 3 weeks, compared with control group, the physical signs such as slim shape, dull action, yellow fur, white ears, nose, slip and tongue had statistical significance; Blood glucose and hemorheology increased significantly; the body weight and strength of grabbing decreased significantly, the intake of water and food, the volume of urine and body temperature did not have statistical significance. In the latter 3 weeks, sticky and loose stool and heavy-red-color tongue increased significantly; After 6 weeks of oral intake of formulas, compared with model group, the physical signs had statistical significance, such as slim shape, dull action, white ears, nose, slip and tongue, especially in the latter 3 weeks. The blood glucose, blood lipid, liver function and renal function also had statistical significance; Compared with the same group in the former 3 weeks, the metabolites in urine of model group had statistical significance, including dihydroxybutanoic acid, L-ascorbic acid, D-gluconic acid, octadecanoic acid, pentanedioic acid. Compared with the same group in the former 3 weeks, L-ascorbic acid had decreased significantly in the latter 3 weeks in the formulas group.
Conclusion:The physical-sign-collecting form could be a suitable instrument for collecting physical signs totally. The physical signs of diabetic rat models built up with STZ are stable through the 3rd to 7th weeks. The syndrome can be diagnosed as the syndrome of stomach heat at first and spleen deficiency at latter, but the syndrome is not found commonly in clinics; the physical signs of diabetic rat models induced by high fat diet and STZ are stable through the 1st to 6th weeks. The syndrome of rat models may be diagnosed as the syndrome of spleen deficiency and intermingled phlegm and blood stasis, which is often found in clinics. The kind of rat models may be qualified for research of verifying syndrome by corresponding formulas; the formulas showed its effects on the physical signs and laboratory items. The evaluation of curative effects could be carried out by estimating physical signs and changes of laboratory items; the changes of metabolites in urine may explain the mechanism of syndrome and curative effects of formulas.
方法：实验研究一即腹腔注射链脲佐菌素诱发糖尿病大鼠模型,初步建立病证结合糖尿病大鼠模型的表征采集表,检测阳性表征及血糖、激素、代谢笼、体温、体重等指标,以临床证候诊断标准及文献报道的相关专家观点为大鼠证候属性的判断依据,对糖尿病大鼠进行初步的证候属性判断；实验研究二即高脂饲料叠加链脲佐菌素复制2型糖尿病大鼠模型,根据动物实验一所建立的表征采集表对大鼠模型进行表征采集,检测血糖、血脂、血流变及激素等指标,结合上述结果判断大鼠模型的证候属性；实验研究三根据方证相应理论,采用具有补脾益气、祛痰化瘀功效的中药复方(高、中、低三个剂量组)干预2型糖尿病大鼠,检测血糖、血脂、胰腺和肝脏病理变化等一般理化指标,结合阳性表征结果,初步评价中药复方的疗效；实验研究四采用气质联用技术对2型糖尿病大鼠(中药高剂量组、西药组、模型组、对照组)的尿液进行检测,根据尿液代谢物的结果,阐释不同组、前后两个时间点(时间点一代表脾气亏虚证为主的阶段,时间点二代表痰湿瘀滞证为主的阶段)大鼠模型的证候及其演变规律的相关生物学基础,以及中药复方干预大鼠模型的疗效机制。
结果：实验研究一建立并优化了实验性糖尿病大鼠的表征采集表,制定了操作简便的表征采集方法；实验研究二以临床证候诊断标准及文献报道的相关专家观点为依据,以业已建立的采集表征表为主要工具,初步建立了病证结合2型糖尿病大鼠的证候属性判断方法。实验研究二建立的2型糖尿病大鼠模型,第1-3周模型组大鼠表征出现形体消瘦、行为呆滞、毛色黄、耳、鼻、唇、舌、爪颜色淡白和尾部重度角化,抓力下降及体质量减低,但饮食量、饮水量、尿量、体温与对照组相比没有差别。第4-6周模型组大鼠部分表征发生进一步变化,与前3周相比,上述阳性表征的比率有上升趋势,并且舌色暗红及大便黏滞松软比率明显增加；实验研究三建立的2型糖尿病大鼠模型经过中药复方干预6周后,一般理化指标和阳性表征均有不同程度的改善。中药高剂量组大鼠模型的血糖、胆固醇、甘油三酯、低密度脂蛋白、尿素氮、谷丙转氨酶和谷草转氨酶水平明显降低。在中药复方改善大鼠模型表征方面,中药高剂量组大鼠的行为活跃比率、耳鼻唇爪颜色淡白比率、舌暗红比率、以及大便黏滞松软比率均有明显改善,出现表征改善的时间点多在中药复方干预的中后期；实验研究四尿液代谢物鉴定结果显示,与时间点一比较,时间点二模型组大鼠尿液中二羟基苯甲酸、L-抗坏血酸、D-葡萄糖酸十八烷酸和戊二酸含量均明显增加；时间点二中药高剂量组大鼠的尿液中L-抗坏血酸含量减少,
结论：实验研究一制定的表征采集表可全面、动态地采集糖尿病大鼠的宏观表征,大鼠成模后第3-7周的表征基本稳定,结合一般理化指标,初步推测模型组大鼠证候特征为早期胃热炽盛证为主,后期脾气亏虚证为主。实验研究一大鼠的证候属性尚不属于文献报道的糖尿病临床常见证型范畴；实验研究二结合大鼠阳性表征和一般理化指标,发现2型糖尿病大鼠第1-6周的表征基本稳定,初步推测模型组大鼠证候特征为前期脾气亏虚证为主,后期痰湿瘀滞证为主。实验研究二大鼠的证候属性属于糖尿病临床常见证型范畴,可作为方证相应研究的合格载体；实验研究三显示高剂量中药复方具有改善2型糖尿病大鼠表征和一般理化指标的作用,从方证相应的角度证明了2型糖尿病大鼠的证候属性可能为脾气亏虚、痰湿瘀滞证。笔者认为从表征和一般理化指标两个方面对2型糖尿病大鼠进行证候疗效评价具有可行性；实验研究四显示,大鼠尿液代谢物的变化可在一定程度上揭示病证结合2型糖尿病大鼠的证候相关生物学基础及中药药效的机制。
Objectives:To establish the physical-sign-collecting form and its collection methods; To explore the method of estimating the syndrome of animal models combining disease with syndrome of Type 2 DM, which are duplicated with high-fat diet and Streptozotocin (STZ); To evaluate the curative effect of formulas on animal models combining disease with syndrome of Type 2 DM; To explore the essence of syndrome of animal models and the mechanism of curative effect of formulas by means of urine metabonomics.
Methods:The research on physical signs and syndrome of diabetic rat models built up with STZ was carried out, the physical-sign-collecting form and its collection method were established. Blood glucose, blood lipid, hormone, body temperature, body weight and so on were measured. The diagnosis of syndrome of rat models was made in the basis of combination of diagnostic standard of clinical syndrome and viewpoints of specialists; The research on physical signs and syndrome of diabetic rat models built up with high-fat diet and STZ was carried out. The physical signs were collected according to the former physical-sign-collecting form. Blood glucose, blood lipid, hormone and so on were measured. The diagnosis of syndrome of rat models was made in the same method; The research on evaluating the curative effect of formulas applied on rat models built up with high-fat diet and STZ was carried out. The estimation of curative effect of formulas was made by means of physical-sign-collection and measurement of blood glucose, blood lipid and pathological changes; The research on applying GC-MS to analyzing the urine of formula-treating rat models built up with high-fat diet and STZ was carried out. The differences of metabolites in different groups and time were analyzed and explained the biological basis of syndrome and mechanism of curative effect.
Results:The physical-sign-collecting form and its collection method were established through the research on physical signs and syndrome of diabetic rat models built up with STZ; On the basis of collecting physical signs and measuring blood, the method of diagnosing syndrome of rat models built up with high-fat diet and STZ had been established according to the combination of diagnostic standard of clinical syndrome and viewpoints of specialists. In the former 3 weeks, compared with control group, the physical signs such as slim shape, dull action, yellow fur, white ears, nose, slip and tongue had statistical significance; Blood glucose and hemorheology increased significantly; the body weight and strength of grabbing decreased significantly, the intake of water and food, the volume of urine and body temperature did not have statistical significance. In the latter 3 weeks, sticky and loose stool and heavy-red-color tongue increased significantly; After 6 weeks of oral intake of formulas, compared with model group, the physical signs had statistical significance, such as slim shape, dull action, white ears, nose, slip and tongue, especially in the latter 3 weeks. The blood glucose, blood lipid, liver function and renal function also had statistical significance; Compared with the same group in the former 3 weeks, the metabolites in urine of model group had statistical significance, including dihydroxybutanoic acid, L-ascorbic acid, D-gluconic acid, octadecanoic acid, pentanedioic acid. Compared with the same group in the former 3 weeks, L-ascorbic acid had decreased significantly in the latter 3 weeks in the formulas group.
Conclusion:The physical-sign-collecting form could be a suitable instrument for collecting physical signs totally. The physical signs of diabetic rat models built up with STZ are stable through the 3rd to 7th weeks. The syndrome can be diagnosed as the syndrome of stomach heat at first and spleen deficiency at latter, but the syndrome is not found commonly in clinics; the physical signs of diabetic rat models induced by high fat diet and STZ are stable through the 1st to 6th weeks. The syndrome of rat models may be diagnosed as the syndrome of spleen deficiency and intermingled phlegm and blood stasis, which is often found in clinics. The kind of rat models may be qualified for research of verifying syndrome by corresponding formulas; the formulas showed its effects on the physical signs and laboratory items. The evaluation of curative effects could be carried out by estimating physical signs and changes of laboratory items; the changes of metabolites in urine may explain the mechanism of syndrome and curative effects of formulas.
引文
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[1]贾伟,赵立平,陈闽军,等.代谢组学在中医药复杂理论体系研究中的应用[J].中国中药杂志,2006,31(8)：621-624.
[2]Gennan JB, Bauman DE, Burrrin DG, et al.decade of the 21st century: Metabolomics in the opening,building the roads to individualized health[J].J Nutn,2004, 134(10):2729.
[3]Devaux PG, Horning MG, Horning EC.Benyzl-oxime derivative of steroids: a new metabolic pro file procedure for human urinary steroids [J].Anal Lett,1971,4:151.
[4]Oliver S.Yeast as a navigational aid in genome analysis [J].Microbiology,1997, 143:1483.
[5]Nicholson JK, Lindon JC, Holmes E.'Metabonomics':understanding the metabolic responses of living systems to pathophysio logical stimuli via multivariate statistical analysis of biological NMR spectroscopic data [J].Xenbiotica,1999,29:1181-1189.
[6]Nicholson JK, Bollard ME, Lindon JC, et al.Metabonomics:a platform for studying drug toxicity and gene function [J].Nat Rev Drug Discov,2002,1:153-162.
[7]FIEHN O, KOPKA J, DORMANN P, etal.Metabolite profiling for plant functional genomics[J].Nat Biotechnol,2000,18(11):57-61.
[8]罗和古,陈家旭.代谢组学技术与中医证候的研究[J].中国中医药信息杂志,2007,14(5)：3-5.
[9]Nicholson JK, Oflynn MP, Sadler PJ, et al.Proton-nuclear-magnetic-resonance studies of serum, plasma and urine from fasting normal and diabetic subjects [J]. Biochem,1984, 217:365-375.
[10]Zuppi C, Messana I, Forni F, et al. 1H NMR spectra of normal urines reference ranges of the major metabolites [J]. Clinica Chimica Acta,1997,265:85-97.
[11]Messana I, Forni F, Zuppi C, et al. Proton nuclear magnetic resonance spectral profiles of urine in type Ⅱ diabetic patients [J]. Clinical Chemistry,1998,44(7):1529-1534.
[12]韩晓菲,黄宇虹,王龙星,等.血浆氨基酸代谢谱与糖尿病相关性研究[J].分析化学,2010,5(38)：697-701.
[13]Jin ES, Burgess SC, Merritt M, et al. Differing mechanisms of hepatic glucose overproduction in triiodothyronine treated rats vs Zucker diabetic fatty rats by NMR analysis of plasma glucose [J]. Am J Physiol Endocrinal Metab,2005,288:E654-E662.
[14]Dumas ME, Wilder SP, Bihoreau MT, et al. Direct quantitative trait locus mapping of mammalian metabolic pheno types in diabetic and normal glycemic rat models [J]. NATURE GENETICS,2007,39(5):666-672.
[15]Toye AA, Dumas ME, Blancher C, et al. Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice [J].Diabetologia,2007,50:1867-1879.
[16]Williams RE, Lenz EM, Evans JA, et al. The comparative metabonomics of age-related changes in the urinary composition of male Wistar-derived and Zucker (fa/fa) obese rats [J]. Molecular Biosystems,2006,2(3-4):193-202.
[17]张淑香,孙晖,孙文军,等.基于临床化学及代谢组学的实验性2型糖尿病大鼠模型评价研究[J].中国中医药现代远程教育,2010,8(10)：1.
[18]蔡爽,霍韬光,徐静华,等.超高效液相色谱-质谱用于2型糖尿病大鼠粪样代谢物指纹图谱研究[J].沈阳药科大学学报,2009,26(10)：811-817.
[19]Nicholson JK, Holmes E, Lindon JC, et al. The challenges of modeling mammalian biocomplexity [J].Nat Biotech,2004,22 (10):1268-1274.
[20]孙永宁,刘忠,冯雯.糖尿病肾阴虚证模型大鼠尿液的代谢组学研究[J].成都中医药大学学报,2009,32(2)：69-71.
[21]孙永宁,冯雯,刘忠.糖尿病肾阴虚证模型大鼠血液标本的代谢组学研究[J].山东中医药大学学报,2010,34(1)：80-82.
[22]和红兵,石先哲,陈静,等.云南白药对实验性糖尿病牙周炎大鼠的临床及代谢组学研究[J].世界科学技术,2009,11(1)：195-201.
[23]王广基,查伟斌,郝海平,等.代谢组学技术在中医药关键科学问题研究中的应用前景分析[J].中国天然药物,2008,6(2)：89-91.
[24]陈蔚文.证候研究要把握证候的本质特征[J].中国中西医结合杂志,2002,22(6)：409-410.[25]罗和古,陈家旭.代谢组学技术与中医证候的研究[J].中国中医药信息杂志,2007,14(5)：3-5.
[1]张业.2型糖尿病胰岛素抵抗大鼠模型证候演变与相关方药作用研究[D].2009
[2]钱俊文,柴可夫.胰岛素抵抗模型形成过程的中医证候动态演变研究[J].中华中医药杂志.2006,(7)：47-49.
[3]Ulupi S. Jhala, Gianluca Canettieri, Robert A. Screaton, etc. cAMP promotes pancreatic-cell survival via CREB-mediated induction of IRS2 [J]. GENES & DEVELOPMENT.2003,17:1575-1580.
[4]冯建华,姜国胜,徐云生,等.化痰活血法改善2型糖尿病模型大鼠胰岛素抵抗的作用机制研究[J].中华中医药杂志.2009,24(8)：1014-1020.
[5]刘小美,方肇勤,卢文丽,等.糖尿病模型大鼠证候的特征及演变[J].辽宁中医杂志,2008,35(1)：130-134.
[6]陈奇.中药药效研究思路与方法.人民卫生出版社.2005：620.
[7]朱文锋.中医诊断学.上海：上海科学技术出版社,1995：149.
[1]潘秋,韩静,余俊达,等.STZ诱发性糖尿病大鼠表征及其证候特征研究[J].中华中医药杂志,2010,25(10)：1644-1647
[2]MJ Reed, K Meszaros, L Entes, et al.A New Rat Model of Type 2 Diabetes:The Fat-Fed, Streptozotocin-Treated Rat[J]. Metabolism,2000,49, pp:1390-1394
[3]冯建华,姜国胜,徐云生,等.化痰活血法改善2型糖尿病模型大鼠胰岛素抵抗的作用机制研究[J].中华中医药杂志,2009,24(8)：1014-1020
[4]刘小美,方肇勤,卢文丽,等.糖尿病模型大鼠证候的特征及演变[J].辽宁中医杂志,2008,35(1)：130-134
[5]朱文锋.中医诊断学.上海：上海科学技术出版社,1995：149
[1]李建生,余学庆,李素云.病证结合诊疗模式下实现证候疗效评价价值的可行途径[J].中华中医药杂志,2009,24(3)：261-264
[2]张艳宏,刘保延,刘志顺,等.PRO与中医临床疗效评价[J].中医杂志,2007,48(8)：680-682
[3]赖世隆.中医药临床疗效的评价[J].中国中医药信息杂志,2000,7(3)：88-89
[4]潘志强,方肇勤,章怡祎.行为学检测在实验小鼠辨证中的应用[J].上海中医药大学学报,2007,21(1)：33-36
[5]方肇勤,潘志强,陈晓.实验小鼠四诊方法学的创建和意义[J].上海中医药杂志,2006,40(7)：1-4
[1]简维雄,黄献平,陈清华,等.基于气相色谱-质谱的大鼠心血瘀阻证血浆代谢组学研究[J].中华中医药学刊,2009,27(4)：796-798
[2]简维雄,袁肇凯,黄献平,等.冠心病心血瘀阻证尿液代谢组学的检测分析[J].中医杂志,2010,51(8)：729-732
[3]孙永宁,冯雯,刘忠.糖尿病肾阴虚证模型大鼠血液标本的代谢组学研究[J].2010,34(1)：80-82
[2]杨彩凤,曹茜,沈永明.2型糖尿病中医证候研究进展[J].天津中医药大学学报,2008,27(2)：106-109.
[3]崔轶凡,王庆国.证候动物模型对中医药研究的意义及方法学探讨[J].中华中医药学刊,2009,27(12)：2525-2527.
[4]李敬林,王太一,王禄增,等.2型糖尿病证病结合动物模型的研究[J].中国比较医学杂志,2007 17(8)：473-475.
[5]苗明三,张玉林,杨保新.病证方结合研究的思路[J].时珍国医国药2008,19(1)：95-96.
[6]支勇,曹式丽.阿霉素肾病动物模型的国外研究进展[J].中国中西结合肾病杂志,2008,9(10)：933-935.
[7]杨进,陆平成,龚婕宁.表证动物模型研制的思路与方法[J].中国中医药科技,1996,3(1)：35-36.
[8]王志刚,岳辉.丹参对实验性2型糖尿病大鼠肾损害的保护作用[J].牡丹江医学院学报,2005,26(1)： 20-21.
[9]潘秋,韩静,余俊达,等.STZ诱发性糖尿病大鼠表征及其证候特征研究[J].中华中医药杂志,2010,25(10)：1644-1647.
[10]邱乐.寒凝血瘀型2型糖尿病动物模型的研究[J].辽宁中医学院硕士学位论文.2003.5.
[11]潘秋,赵慧辉,陈建新,等.2型糖尿病实验大鼠表征及其证候特征研究[J].中华中医药杂志,2011,26(4)：683-685.
[12]康洁,高碧珍.病证结合动物模型研究概况[J].中华中医药学刊,2009,27(11)：2357-2359.
[13]方肇勤.辨证论治研发的一些基本学术问题[J].中国中西医结合杂志,2008,28(7)：652-655.
[14]富琦,陈信义.建立病证结合动物模型的新思路[J].中国中医药信息杂志,2003,10(9)：79.
[15]郑方遒,李敬林,刘伟.2型糖尿病气阴两虚证病证结合模型的研究[J].中华中医药杂志,2009,24(5)：593-595.
[16]冯雯,孙永宁.糖尿病阴虚证动物模型的复制[J].安徽中医学院学报,2009,28(2)： 40-42.
[17]董天宝.阴虚热盛型2型糖尿病动物模型的初步研究[J].实用中医内科杂志,2009,3(26)： 31-33.
[18]朱章志,黄从强,何敏.健脾益气中药对脾气虚证2型糖尿病大鼠胰岛素介体的影响[J].中药新药与临床药理,2004,15(2)：107-109.
[19]王阶,荆鲁,衷敬柏.血府逐淤汤拆方临床研究[J].中国中药杂志,2004,29(8)：803.
[20]陈小野.证候动物模型诊断依据的设想与评价[J].中国医药学报,1987,2(1)：50-53.
[21]赵慧辉,郭书文,王伟.病证结合动物模型判定标准的建立[J].北京中医药大学学报,2009,32(6)：365-368.
[1]贾伟,赵立平,陈闽军,等.代谢组学在中医药复杂理论体系研究中的应用[J].中国中药杂志,2006,31(8)：621-624.
[2]Gennan JB, Bauman DE, Burrrin DG, et al.decade of the 21st century: Metabolomics in the opening,building the roads to individualized health[J].J Nutn,2004, 134(10):2729.
[3]Devaux PG, Horning MG, Horning EC.Benyzl-oxime derivative of steroids: a new metabolic pro file procedure for human urinary steroids [J].Anal Lett,1971,4:151.
[4]Oliver S.Yeast as a navigational aid in genome analysis [J].Microbiology,1997, 143:1483.
[5]Nicholson JK, Lindon JC, Holmes E.'Metabonomics':understanding the metabolic responses of living systems to pathophysio logical stimuli via multivariate statistical analysis of biological NMR spectroscopic data [J].Xenbiotica,1999,29:1181-1189.
[6]Nicholson JK, Bollard ME, Lindon JC, et al.Metabonomics:a platform for studying drug toxicity and gene function [J].Nat Rev Drug Discov,2002,1:153-162.
[7]FIEHN O, KOPKA J, DORMANN P, etal.Metabolite profiling for plant functional genomics[J].Nat Biotechnol,2000,18(11):57-61.
[8]罗和古,陈家旭.代谢组学技术与中医证候的研究[J].中国中医药信息杂志,2007,14(5)：3-5.
[9]Nicholson JK, Oflynn MP, Sadler PJ, et al.Proton-nuclear-magnetic-resonance studies of serum, plasma and urine from fasting normal and diabetic subjects [J]. Biochem,1984, 217:365-375.
[10]Zuppi C, Messana I, Forni F, et al. 1H NMR spectra of normal urines reference ranges of the major metabolites [J]. Clinica Chimica Acta,1997,265:85-97.
[11]Messana I, Forni F, Zuppi C, et al. Proton nuclear magnetic resonance spectral profiles of urine in type Ⅱ diabetic patients [J]. Clinical Chemistry,1998,44(7):1529-1534.
[12]韩晓菲,黄宇虹,王龙星,等.血浆氨基酸代谢谱与糖尿病相关性研究[J].分析化学,2010,5(38)：697-701.
[13]Jin ES, Burgess SC, Merritt M, et al. Differing mechanisms of hepatic glucose overproduction in triiodothyronine treated rats vs Zucker diabetic fatty rats by NMR analysis of plasma glucose [J]. Am J Physiol Endocrinal Metab,2005,288:E654-E662.
[14]Dumas ME, Wilder SP, Bihoreau MT, et al. Direct quantitative trait locus mapping of mammalian metabolic pheno types in diabetic and normal glycemic rat models [J]. NATURE GENETICS,2007,39(5):666-672.
[15]Toye AA, Dumas ME, Blancher C, et al. Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice [J].Diabetologia,2007,50:1867-1879.
[16]Williams RE, Lenz EM, Evans JA, et al. The comparative metabonomics of age-related changes in the urinary composition of male Wistar-derived and Zucker (fa/fa) obese rats [J]. Molecular Biosystems,2006,2(3-4):193-202.
[17]张淑香,孙晖,孙文军,等.基于临床化学及代谢组学的实验性2型糖尿病大鼠模型评价研究[J].中国中医药现代远程教育,2010,8(10)：1.
[18]蔡爽,霍韬光,徐静华,等.超高效液相色谱-质谱用于2型糖尿病大鼠粪样代谢物指纹图谱研究[J].沈阳药科大学学报,2009,26(10)：811-817.
[19]Nicholson JK, Holmes E, Lindon JC, et al. The challenges of modeling mammalian biocomplexity [J].Nat Biotech,2004,22 (10):1268-1274.
[20]孙永宁,刘忠,冯雯.糖尿病肾阴虚证模型大鼠尿液的代谢组学研究[J].成都中医药大学学报,2009,32(2)：69-71.
[21]孙永宁,冯雯,刘忠.糖尿病肾阴虚证模型大鼠血液标本的代谢组学研究[J].山东中医药大学学报,2010,34(1)：80-82.
[22]和红兵,石先哲,陈静,等.云南白药对实验性糖尿病牙周炎大鼠的临床及代谢组学研究[J].世界科学技术,2009,11(1)：195-201.
[23]王广基,查伟斌,郝海平,等.代谢组学技术在中医药关键科学问题研究中的应用前景分析[J].中国天然药物,2008,6(2)：89-91.
[24]陈蔚文.证候研究要把握证候的本质特征[J].中国中西医结合杂志,2002,22(6)：409-410.[25]罗和古,陈家旭.代谢组学技术与中医证候的研究[J].中国中医药信息杂志,2007,14(5)：3-5.
[1]张业.2型糖尿病胰岛素抵抗大鼠模型证候演变与相关方药作用研究[D].2009
[2]钱俊文,柴可夫.胰岛素抵抗模型形成过程的中医证候动态演变研究[J].中华中医药杂志.2006,(7)：47-49.
[3]Ulupi S. Jhala, Gianluca Canettieri, Robert A. Screaton, etc. cAMP promotes pancreatic-cell survival via CREB-mediated induction of IRS2 [J]. GENES & DEVELOPMENT.2003,17:1575-1580.
[4]冯建华,姜国胜,徐云生,等.化痰活血法改善2型糖尿病模型大鼠胰岛素抵抗的作用机制研究[J].中华中医药杂志.2009,24(8)：1014-1020.
[5]刘小美,方肇勤,卢文丽,等.糖尿病模型大鼠证候的特征及演变[J].辽宁中医杂志,2008,35(1)：130-134.
[6]陈奇.中药药效研究思路与方法.人民卫生出版社.2005：620.
[7]朱文锋.中医诊断学.上海：上海科学技术出版社,1995：149.
[1]潘秋,韩静,余俊达,等.STZ诱发性糖尿病大鼠表征及其证候特征研究[J].中华中医药杂志,2010,25(10)：1644-1647
[2]MJ Reed, K Meszaros, L Entes, et al.A New Rat Model of Type 2 Diabetes:The Fat-Fed, Streptozotocin-Treated Rat[J]. Metabolism,2000,49, pp:1390-1394
[3]冯建华,姜国胜,徐云生,等.化痰活血法改善2型糖尿病模型大鼠胰岛素抵抗的作用机制研究[J].中华中医药杂志,2009,24(8)：1014-1020
[4]刘小美,方肇勤,卢文丽,等.糖尿病模型大鼠证候的特征及演变[J].辽宁中医杂志,2008,35(1)：130-134
[5]朱文锋.中医诊断学.上海：上海科学技术出版社,1995：149
[1]李建生,余学庆,李素云.病证结合诊疗模式下实现证候疗效评价价值的可行途径[J].中华中医药杂志,2009,24(3)：261-264
[2]张艳宏,刘保延,刘志顺,等.PRO与中医临床疗效评价[J].中医杂志,2007,48(8)：680-682
[3]赖世隆.中医药临床疗效的评价[J].中国中医药信息杂志,2000,7(3)：88-89
[4]潘志强,方肇勤,章怡祎.行为学检测在实验小鼠辨证中的应用[J].上海中医药大学学报,2007,21(1)：33-36
[5]方肇勤,潘志强,陈晓.实验小鼠四诊方法学的创建和意义[J].上海中医药杂志,2006,40(7)：1-4
[1]简维雄,黄献平,陈清华,等.基于气相色谱-质谱的大鼠心血瘀阻证血浆代谢组学研究[J].中华中医药学刊,2009,27(4)：796-798
[2]简维雄,袁肇凯,黄献平,等.冠心病心血瘀阻证尿液代谢组学的检测分析[J].中医杂志,2010,51(8)：729-732
[3]孙永宁,冯雯,刘忠.糖尿病肾阴虚证模型大鼠血液标本的代谢组学研究[J].2010,34(1)：80-82