“肾阳虚”模型及证候的代谢组学研究
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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.
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
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