节点文献
阳虚体质、阴虚体质与脾胃虚寒证、胃阴亏虚证的代谢组学比较研究
【作者】 袁卓珺；
【导师】 王琦；
【作者基本信息】 北京中医药大学 ， 中医基础理论， 2011， 博士
【摘要】 目的：挖掘中医学中阳虚、阴虚证候及阳虚、阴虚体质的相关内容,总结概述近现代研究进展及成果,进一步充实中医体质与证候的关系研究；比较研究脾胃阳虚证、胃阴亏虚证及其与阳虚体质、阴虚体质的代谢组学(metabonomics)特征,探索证候与体质分类的依据及形成机制。为阐述体质与证候的关系提供生物分子学依据。方法：采用传统文献梳理和现代文献分析的方法,进行阳虚、阴虚证候及阳虚、阴虚体质的理论研究。运用1H-NMR技术,采用美国瓦里安公司(Varian,Inc.) INOVA 600 MHz超导核磁共振谱仪,比较分析脾胃阳虚证、胃阴亏虚证及阳虚、阴虚体质血液和尿液的代谢组学特征,寻找生物标志物。结果：阳虚、阴虚体质与脾胃虚寒证、胃阴亏虚证的代谢组学特征研究,通过比较每组30例的代谢产物谱特征,以健康人作为对照,结合理论研究和以往的实验研究结果发现：①代谢组学的方法能够对证候进行客观分类。与健康人相比,脾胃虚寒证、胃阴亏虚证存在代谢网路的改变；②代谢组学方法能够阐释体质与证候的联系与区别的关系问题。与脾胃虚寒证、胃阴亏虚证相比,阳虚体质、阴虚体质存在代谢网路的改变；③发现脾胃虚寒证、胃阴亏虚证的潜在生物标志物,并明确了含量的变化方向。脾胃虚寒证血液中的标记物有乳酸(lactate,δ1.32,1.33)、低密度脂蛋白(LDL,δ0.88,0.89,1.22,1.26,1.27～1.29)、丙氨酸(Ala,δ1.47,1.48)、高密度脂蛋白(HDL,δ0.85,0.86)、葡萄糖(glucose,δ3.4～3.7)、甘氨酸(Gly,δ3.55),极低密度脂蛋白(VLDL,δ0.94,1.38,1.34),不饱和脂肪酸(UFA,65.3)、磷脂酰胆碱(Ptd,δ3.22)。胃阴亏虚证血液中的标记物有乳酸(lactate,δ1.32,1.33)、低密度脂蛋白(LDL,δ0.88,0.89,1.22,1.26,1.27-1.29)、丙氨酸(Ala,δ1.47,1.48)、高密度脂蛋白(HDL,δ0.85,0.86)、葡萄糖(glucose,δ3.4～3.7)、甘氨酸(Gly,63.55)、极低密度脂蛋白(VLDL,δ0.94,1.38,1.34),不饱和脂肪酸(UFA,δ5.22,5.3,5.34),磷脂酰胆碱(Ptd,δ3.22)。其中,与健康人相比,脾胃虚寒证血液中乳酸(lactate,δ1.32,1.33),低密度脂蛋白(LDL,δ0.88,0.89,1.27-1.29),丙氨酸(Ala,δ1.47,1.48),葡萄糖(glucose,δ3.4～3.7),甘氨酸(Gly,δ3.55)含量相对较低。高密度脂蛋白(HDL,δ0.85,0.86),低密度脂蛋白(LDL,61.22,1.26),极低密度脂蛋白(VLDL,δ0.94,1.38,1.34),不饱和脂肪酸(UFA,δ5.3)、磷脂酰胆碱(Ptd,δ3.22)含量相对较高。胃阴亏虚证血液中乳酸(lactate,δ1.32,1.33),低密度脂蛋白(LDL,δ0.88,0.89,1.27～1.29)、丙氨酸(Ala,61.47,1.48)、高密度脂蛋白(HDL,δ0.85,0.86)、甘氨酸(Gly,63.55)含量相对较低。高密度脂蛋白(HDL,δ0.86),低密度脂蛋白(LDL,61.22,1.26),极低密度脂蛋白(VLDL,δ0.94,1.38,1.34),不饱和脂肪酸(UFA,δ5.22,5.3,5.34),磷脂酰胆碱(Ptd,63.22)含量相对较高。葡萄糖(glucose,δ3.4～3.7)含量相对较低。胃阴亏虚证与脾胃虚寒证相比,血液中乳酸(lactate,δ1.32,1.33),低密度脂蛋白(LDL,δ0.88,0.89,1.26-1.29),丙氨酸(Ala,δ1.48),高密度脂蛋白(HDL,50.85,0.86),甘氨酸(Gly,δ3.55),极低密度脂蛋白(VLDL,δ1.3,1.34),不饱和脂肪酸(UFA,δ5.3)含量相对较低。高密度脂蛋白(HDL,δ0.86),葡萄糖(glucose,δ3.4～3.7),磷脂酰胆碱(Ptd,δ3.22)含量相对较高。脾胃亏虚证与阳虚体质相比,脾胃虚寒证组血浆中丙氨酸(Ala,δ1.48),谷氨酰胺(Gln,62.12,2.13,2.45,2.46,3.74)、甘氨酸(Gly,δ3.55)、不饱和脂肪酸(UFA,δ5.22,5.3,5.34)的含量相对减少。而葡萄糖(glucose,δ3.4～3.7)、高密度脂蛋白(HDL,δ0.86),低密度脂蛋白(LDL,δ0.9)、极低密度脂蛋白(VLDL,δ0.94,1.38).N-乙酰糖蛋白(Nac,δ2.02)、磷脂酰胆碱(Ptd,δ3.22)的含量相对升高。胃阴亏虚证与阴虚体质相比,胃阴亏虚证组血浆中乳酸(Lac,δ1.32,1.33),低密度脂蛋白(LDL,δ0.89,1.18,1.28)、谷氨酰胺(Gln,δ2.45,2.46)、饱和脂肪酸(FA,δ2.22)、不饱和脂肪酸(UFA,δ5.3,5.34)的含量均相对下降；而丙氨酸(Ala,61.46～1.48)、葡萄糖(glucose,δ3.4～3.7)、甘氨酸(Gly,δ3.54)、高密度脂蛋白(HDL,δ0.86),极低密度脂蛋白(VLDL,δ0.94,1.38,1.34)、N-乙酰糖蛋白(Nac,δ2.02)、磷脂酰胆碱(Ptd,63.22)的含量相对升高。脾胃虚寒证尿液中的标记物有：乳酸(lactate,δ1.34,4.14,4.18)、丙酮酸盐(pyeuvate,δ2.38)、柠檬酸(citric acid,62.54,2.66,2.70)、二甲胺(dimethylamine,δ2.74)、肌酐(creatinine,δ3.06,4.06),氧化三甲胺(Trimetlylamine xide,δ3.26),牛磺酸(taurine,δ3.26,3.42).甘氨酸(Gly,δ3.58)、马尿酸(hippuric acid,δ3.98,7.54,7.58,7.62, 7.66,7.82,7.86)。胃阴亏虚证尿液中的标记物有：乳酸(lactate,δ1.34,4.14,4.18)、丙酮酸盐(pyeuvate,62.38)、柠檬酸(citric acid,δ2.54,2.66,2.70)、二甲胺(dimethylamine,δ2.74)、肌酐(creatinine,δ3.06,4.06),氧化三甲胺(Trimetlylamine xide,δ3.26),牛磺酸(taurine,δ3.26,3.42).甘氨酸(Gly,δ3.58)、马尿酸(hippuric acid,δ3.98,7.54, 7.58,7.62,7.66,7.82,7.86)。其中,与健康人相比,脾胃虚寒证、胃阴亏虚证尿液中乳酸(lactate,δ1.34,4.14,4.18)、柠檬酸(citric acid,δ2.54,2.66,2.70).二甲胺(dimethylamine,δ2.74).肌酐(creatinine,δ3.06,4.06),氧化三甲胺(Trimetlylamine xide,δ3.26),甘氨酸(Gly,δ3.58)含量相对较高。丙酮酸盐(pyeuvate,δ2.38)含量相对较低。脾胃虚寒证与胃阴亏虚证相比,尿液中乳酸(lactate,δ1.34,4.14,4.18),柠檬酸(citric acid,62.54,2.66,2.70)、氧化三甲胺(rimetlylamine xide,δ3.26)、牛磺酸(taurine,δ3.26,3.42)、甘氨酸(Gly,δ3.58)含量相对较高。丙酮酸盐(pyeuvate,δ2.38)、二甲胺(dimethylamine,δ2.74)、肌酐(creatinine,δ3.06,4.06)、马尿酸(hippuric acid,δ3.98,7.54,7.58,7.62,7.66,7.82,7.86)含量相对较低。与阳虚体质相比,脾胃虚寒证组尿液中乳酸(lactate,δ1.34,4.14,4.18),肌酐(creatinine,δ3.06,4.06)、氧化三甲胺(rimetlylamine xide,82.94)、甘氨酸(glycine,3.58)的含量相对升高；而丙酮酸盐(pyeuvate,82.38)的含量相对降低。同时,脾胃虚寒证组与阳虚体质组尿液中在相同化学位移处找到相同标记物,并且组间没有含量差异。柠檬酸(citric acid,82.54,2.66,2.70).二甲胺(dimethylamine,δ2.74)、牛磺酸(taurine,δ3.26,3.42)、马尿酸(hippuric acid,δ3.98,7.54,7.58, 7.62,7.66,7.82,7.86)与阴虚体质相比,胃阴亏虚证组尿液中乳酸(lactate,δ1.34,4.14,4.18),柠檬酸(citric acid,δ2.54,2.66,2.70)、肌酐(creatinine,δ3.06,4.06)、氧化三甲胺((rimetlylamine xide,δ2.94)、甘氨酸(glycine,3.58)的含量相对升高；而丙酮酸盐(pyeuvate,δ2.38)的含量相对降低。同时,胃阴亏虚证组与阴虚体质组尿液中在相同化学位移处找到相同标记物,并且组间没有含量差异。二甲胺(dimethylamine,δ2.74)、牛磺酸(taurine,δ3.26,3.42)、马尿酸(hippuric acid,83.98,7.54,7.58,7.62,7.66,7.82,7.86)。④通过查阅相关数据库和文献,对生物标记物的生理功能和代谢途径进行阐述。发现阳虚、阴虚体质与脾胃虚寒证、胃阴亏虚证在能量代谢、脂代谢、糖代谢、氨基酸代谢方面的差异,还存在神经递质、脏腑功能的改变。证候组较体质组总体表现为代谢功能活动低下。应用代谢组学的技术与方法对阳虚、阴虚体质、脾胃虚寒证、胃阴亏虚证的生物特征进行了探索,可能为体质与证候的联系与区别提供新的依据。结论：通过对阳虚、阴虚体质与证候的理论梳理与比较,全面系统地阐述了二者的主体内涵、形成原因、表现特征、易发病证等方面存在的联系与区别,为实验研究奠定了理论基础。阳虚、阴虚体质与脾胃虚寒证、胃阴亏虚证的差异代谢物的发现,从代谢组学角度为体质与证候的关系提供了客观依据。阳虚、阴虚体质与脾胃虚寒证、胃阴亏虚证存在能量代谢、脂代谢、糖代谢、氨基酸代谢的差异,并伴随着神经递质、脏腑功能的改变。
【Abstract】 Objectives:to study the syndromes of yang deficiency and yin deficiency, and the constitutions of yang deficiency and yin deficiency in Chinese medicine, summarize the research progress and achievements, and further enrich the relationship between constitutions and syndromes; to compare the characteristics between syndromes of spleen and stomach yang deficiency and stomach yin deficiency, and the metabonomics of the constitutions of yang deficiency and yin deficiency, to explore the evidence of the classification of syndromes and constitutions and its mechanism; to provide the molecular biological evidence for the relationship between constitutions and syndromes.Methods:The theory of both the syndromes and constitutions of yang deficiency were studied via methods of literature searching and modern literature analysis. In order to find the biomarker, the characteristics of the metabonomics of the blood and urine in the syndromes of spleen and stomach yang deficiency and stomach yin deficiency and the constitutions of yang deficiency and yin deficiency were compared and analyzed by the technique of 1H-NMR and the Superconducting NMR spectrometer of INOVA 600 MHz (Varian,Inc.)Results:The characteristics of the metabonomics in the syndromes of spleen and stomach yang deficiency and stomach yin deficiency and the constitutions of yang deficiency and yin deficiency were studied. Each group contains 30 samples. The results were combined with the theory and the former experimental study with the control of the normal people:①The metabonomics can classify the syndromes objectively. Compared to the normal people, there were changes of metabolic network in the syndromes of spleen and stomach yang deficiency and stomach yin deficiency;②The metabonomics can explain the relations and differences between the constitutions and syndromes. Compared to the constitutions of yang deficiency and yin deficiency, there were changes of metabolic network in the syndromes of spleen and stomach yang deficiency and stomach yin deficiency;③The potential biomarker of the syndromes of spleen and stomach yang deficiency and stomach yin deficiency was discovered, and the change direction of contents was confirmed.The blood markers of the syndrome of spleen and stomach yang deficiency were lactic acid (lactate,δ1.32,1.33), low density lipoprotein (LDL,δ0.88,0.89,1.22,1.26, 1.27～1.29)、alanine(Ala,δ1.47,1.48), high density lipoprotein(HDL,δ0.85,0.86), glucose (glucose,δ3.4-3.7), glycine (Gly,δ3.55), very low density lipoprotein (VLDL,δ0.94, 1.38,1.34), ultra high tetty acids (UFA,δ5.3)、ultra high tetty acids (Ptd,δ3.22) The blood markers of the syndrome of spleen and stomach yang deficiency were lactic acid (lactate,δ1.32,1.33), low density lipoprotein (LDL,δ0.88,0.89,1.22,1.26, 1.27-1.29), alanine(Ala,δ1.47,1.48),high density lipoprotein(HDL,δ0.85,0.86),glucose (glucose,δ3.4～3.7), glycine (Gly,δ3.55), very low density lipoprotein (VLDL,δ0.94, 1.38,1.34), ultra high tetty acids(UFA,δ5.22,5.3,5.34), ultra high tetty acids(Ptd,δ3.22).Compared to the normal person, lactic acid(lactate,δ1.32,1.33), low density lipoprotein (LDL,δ0.88,0.89,1.27～1.29)、alanine(Ala,δ1.47,1.48), glucose(glucose,δ3.4～3.7), and glycin (Gly,δ3.55) were lower in the blood of spleen and stomach yang deficiency, while high density lipoprotein (HDL,δ0.85,0.86), low density lipoprotein (LDL,δ1.22,1.26), very low density lipoprotein(VLDL,δ0.94,1.38,1.34), ultra high tetty acids(UFA,δ5.3)、phosphatidylcholine (Ptd,δ3.22) expressed higher. The lactic acid (lactate,δ1.32,1.33)、low density lipoprotein (LDL,δ0.88,0.89,1.27-1.29),alanine (Ala,δ1.47,1.48)、high density lipoprotein (HDL,δ0.85,0.86), and glycine (Gly,δ3.55) expressed relatively lower in the syndrome of stomach yin deficiency, while high density lipoprotein (HDL,δ0.86), low density lipoprotein (LDL,δ1.22,1.26), low density lipoprotein (VLDL,δ0.94,1.38, 1.34), low density lipoprotein (UFA,δ5.22,5.3,5.34), phosphatidylcholine (Ptd,δ3.22) expressed relatively higher and glucose (glucose,δ3.4～3.7) was relatively lower.Compared to the syndrome of spleen and stomach yang deficiency, lactic acid (lactate,δ1.32,1.33), low density lipoprotein(LDL,δ0.88,0.89,1.26～1.29), alanine(Ala,δ1.48), high density lipoprotein (HDL,δ0.85,0.86), glycine (Gly,δ3.55), very low density lipoprotein (VLDL,δ1.3,1.34), ultra and high tetty acids (UFA,δ5.3) expressed relatively lower, while high density lipoprotein (HDL,δ0.86), glucose (glucose,δ3.4～3.7), phosphatidylcholine (Ptd,δ3.22) expressed relatively higher in the syndrome of stomach yin deficiency.Compared to the constitution of yang deficiency, the alanine (Ala,δ1.48), glutaminate (Gln,δ2.12,2.13,2.45,2.46,3.74), glycine (Gly,δ3.55). ultra high tetty acids (UFA,δ5.22,5.3,5.34) expressed relatively lower, while the glucose (glucose,δ3.4～3.7), high densitv lipoprotein (HDL,δ0.86), low densitv lipoprotein (LDL,δ0.9),very low densitv lipoprotein(VLDL,δ0.94,1.38), N-acetyl glycoproteins(Nac,δ2.02), phosphatidylcholine (Ptd,δ3.22) expressed relatively higher in the blood of the syndrome of spleen and stomach yang deficiency.Compared to the constitution of yin deficiency, the lactic acid (Lac,δ1.32,1.33), low densitv lipoprotein (LDL,δ0.89,1.18,1.28), glutaminate (Gln,δ2.45,2.46). satisfied fatty acid (FA,δ2.22), ultra high tetty acids (UFA,δ5.3,5.34) expressed relatively lower, while the alanine (Ala,δ1.46～1.48), glucose (glucose,δ3.4～3.7), glycine (Gly,δ3.54), high densitv lipoprotein(HDL,δ0.86), very low densitv lipoprotein (VLDL,δ0.94,1.38, 1.34), N-acetyl glycoproteins(Nac,δ2.02), phosphatidylcholine(Ptd,δ3.22)expressed higher in the blood of the syndrome of stomach yin deficiency.The markers of the urine of the syndrome of spleen and stomach yang deficiency were lactic acid (lactate,δ1.34,4.14,4.18),acetonate; pyruvate (δ2.38),citric acid (δ2.54, 2.66,2.70),dimethylamine(δ2.74),dimethylglycine(δ2.82),trimethylamine(δ2.94),creatinine (δ3.06,4.06),creatinine(δ3.26), creatinine(δ3.26,3.42),glycine(δ3.58),benzoylglycine (δ3.98,7.54,7.58,7.62,7.66,7.82,7.86)The markers of the urine of the syndrome of stomach yin deficiency were:acid(lactate,δ1.34,4.14,4.18),acetonate;pyruvate(δ2.38),citric acid(δ2.54,2.66,2.70),dimethylamine (δ2.74),dimethylglycine (δ2.82),dimethylglycine (δ3.06,4.06), trimethylamine oxide (δ3.26), taurine (δ3.26,3.42),glycine (δ3.58),benzoylglycine (δ3.98,7.54,7.58, 7.62,7.66,7.82,7.86)Compared to the normal people, the lactic acid (lactate,δ1.34,4.14,4.18),lactic acid (δ2.54,2.66,2.70),dimethylamine (δ2.74),creatinine (δ3.06,4.06), trimethylamine oxide (δ3.26),and glycine (δ3.58) expressed relatively higher, while the acetonat (δ2.38) and trimethylamine (δ2.94) expressed relatively lower in the urine of the syndromes of spleen and stomach yang deficiency and stomach yin deficiency.Compared to the syndrome of stomach yin deficiency, the lactic acid (lactate,δ1.34, 4.14,4.18), citric acid (δ2.54,2.66,2.70),trimethylamine (δ2.94),trimethylamine oxide (δ3.26),taurine (δ3.26,3.42),glycine (δ3.58) expressed relatively higher, while the acetonate (δ2.38),dimethylamine (82.74),dimethylamine (δ3.06,4.06),benzoylglycine (δ3.98,7.54,7.58,7.62,7.66,7.82,7.86) expressed relatively lower in the urine of the syndrome of spleen and stomach yang deficiency.Compared to the constitution of yang deficiency, the lactic acid (δ1.34,4.14,4.18), creatinine (creatinine,δ3.06,4.06), trimethylamine oxide (δ2.94), glycine (glycine,3.58) were relatively higher, while the acetonate was relatively lower in the blood of the syndrome of spleen and stomach yang deficiency.The same markers for the syndrome of spleen and stomach yang deficiency and the constitution of yang deficiency in the urine were discovered at the same chemical shift. There was no difference between these two groups. The citric acid (δ2.54,2.66,2.70). dimethylamine (82.74)、trimethylamine (δ2.94). taurine (δ3.26,3.42). taurine (δ3.98, 7.54,7.58,7.62,7.66,7.82,7.86)Compared to the constitution of yin deficiency, the lactic acid(δ1.34,4.14,4.18), citric acid (δ2.54,2.66,2.70), creatinine (creatinine,δ3.06,4.06),trimethylamine oxide (δ2.94),glycine (glycine,3.58) expressed relatively higher, while the pyeuvate (pyeuvateδ2.38) expressed relatively lower in the urine of the syndrome of stomach yin deficiency.The same markers for the syndrome of stomach yin deficiency and the constitution of yin deficiency in the urine were discovered at the same chemical shift. There was no difference between these two groups. The dimethylamine (δ2.74),dimethylglycine (δ2.82),taurine (83.26,3.42),benzoylglycine (δ3.98,7.54,7.58,7.62,7.66,7.82,7.86)④The physiologic function and metabolic pathway was studied by consulting relational database and literature. The differences of energy metabolism, lipid metabolism, glycometabolism, and amino acid metabolism in the syndrome of stomach yin deficiency were discovered with the changes of neurotransmitters and viscera function. The metabolism function of the syndrome group was lower than that of the constitution group.The biological characteristics of the constitutions of yang deficiency and yin deficiency and the syndromes of spleen and stomach yang deficiency and stomach yin deficiency were explored with the techniques and methods of metabonomics, which may provide a novel explanation for the relations and differences between the constitutions and syndromes.Conclusion:The constitutions and the syndromes of yang deficiency and yin deficiency were studied and compared. The relations and differences of connotation, formation reason, manifestation, morbidity, etc were formulated overall, which lays a theory foundation for the experimental study. The discovery of the potential biomarkers of the constitutions of yang deficiency and yin deficiency and the syndromes of spleen and stomach yang deficiency and stomach yin deficiency provides the objective evidence for the relationship between constitutions and syndromes from the aspect of metabonomics. The differences of energy metabolism, lipid metabolism, glycometabolism, and amino acid metabolism between the constitutions of yang deficiency and yin deficiency and the syndromes of spleen and stomach yang deficiency and stomach yin deficiency were discovered with the changes of neurotransmitters and viscera function.