基于阴虚、阳虚体质基因表达的健康状态微观辨识研究
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摘要
研究背景及目的：
深化反映人体相对稳定状态的体质辨识法是建立中国特色健康管理的有效方法。基于体质辨识基因组学研究是获取健康状态微观信息的途径之一。利用全基因组表达谱分析体质对应的分子生物学特征,可以初步把握健康状态的微观生物信息特征。拟选取具有代表性的阳虚体质、阴虚体质两种体质进行分析,有助于揭示这两种体质的生物学特征,有助于健康状态微观辨识研究,同时为区分不同体质的人群提供依据。通过对健康状态微观生物信息的研究,最终为调理阴虚、阳虚体质提供分子生物学依据,为预防其相关性疾病的发生奠定基础,实现有效的全面的全民健康管理体系做出贡献。
方法：
按照阳虚、阴虚体质和平和体质判定标准,并由中医师四诊确认符合,最后筛选出12例阴虚质、12例阳虚体质和8例平和质。取上述受试者的外周血,2小时内分离外周血单个核细胞,提取白细胞中的总RNA.采用Affymetrix3'IVT表达谱芯片,开展cDNA的合成与标记、芯片杂交、芯片的荧光标记与扫描,获取阴虚质、阳虚质、平和质的外周血基因表达谱。进行基因聚类和基因分类器研究,探讨体质分类情况与基因聚类的关系,并探索基因分类器在体质辨识中的应用方法。同时对阴虚体质与阳虚体质的特征和差异表达基因及路通的生物学功能进行评价,探析其健康状态的分子生物学基础。
结果：
1.全部32个样本的所有基因可聚集为三类,这与样本的真实体质分类标签相符合。
2.通过基因分类器找到可区分三种体质的228个探针,并且基因分类器的敏感性、特异性、阳性预测值、阴性预测值四个参数均有意义。
3.当q-value(%)<5%并且Fold Change>2或Fold Change<1/2时,阳虚体质与阴虚体质的基因表达谱获得共232条差异表达基因,其中有223个基因表达水平上调的,有9个基因表达水平下调的。阳虚体质与阴虚体质的差异基因富集到35条差异显著的信号转导通路,主要涉及免疫功能、能量代谢、脂代谢、糖代谢、神经系统以及肿瘤癌症疾病等信号通路。同时阴虚质、阳虚质与平和质对照组比较,二者共同的差异表达基因有60条,其中表达完全相反的有3条,即FFAR2.GPR109B.NAMPT.与阴虚体质相比,阳虚体质表达远远上调,FFAR2(221345_at)Fold Change≈6.7796;GPR109B(205220_at) Fold Change≈7.6858；NAMPT(217739_s_at)Fold Change≈6.1773(P<0.05)。
4.当q-value(%)<5%并且Fold Change>2或者Fold Change<1/2时,阴虚体质组和平和体质对照组间基因表达图谱获得共155条差异表达基因,其中有92条表达水平上调,有63条表达水平下调；阳虚体质组和平和体质对照组间的基因表达图谱获得共203条差异表达基因,其中有51条表达水平上调,有152条基因表达水平下调。阴虚体质的差异表达的基因主要参与免疫应答、炎症反应、细胞凋亡、DNA甲基化、胰岛素分泌调节、钙介导的信号转导、糖代谢、脂质分解代谢、三磷酸腺苷反应等生物过程。差异基因富集到T细胞受体的信号通路、Toll-like受体信号通路、B细胞受体信号通路、Jak-STAT(?)言号通路、丝裂酶原活化蛋白激酶信号转导通路、系统性红斑狼疮、自身免疫性甲状腺疾病、Ⅱ型糖尿病、早老性痴呆及多条癌症等44条信号转导通路。阳虚体质的差异表达基因主要参与免疫应答、炎症反应、信号转导、碳水化合物代谢、调控胰岛素分泌、甘油代谢、脂肪细胞分化、淀粉分解代谢、三磷酸腺苷反应等生物过程。差异基因富集到丝裂酶原活化蛋白激酶信号转导通路、抗原加工提呈通路、tOll样受体信号通路、T细胞受体的信号通路、Wnt信号通路、肽聚糖生物合成、B细胞受体的信号通路、系统性红斑狼疮、哮喘、自身免疫性甲状腺疾病、上皮细胞幽门螺杆菌感染、早老性痴呆、I型糖尿病、膀胱癌、结肠直肠癌等30条信号转导通路。
结论：
1.阴虚体质、阳虚体质和平和体质具有各自体质的基因表达谱特征。
2.中医体质分类与基因分类结果相符合。通过基因分类器方面可以初步区分阴虚体质、阳虚体质和平和体质,同时为健康状态微观辨识研究提供方法学依据。
3.阳虚体质和阴虚体质存在基因表达图谱显著差异。差异基因主要反映阳虚体质与阴虚体质在免疫功能、能量代谢、脂代谢、糖代谢、神经系统以及肿瘤癌症疾病等方面存在很大差别。研究结果表明阳虚体质与阴虚体质在糖脂代谢上存在反向差异,这符合阳虚体质和阴虚体质热量代谢相反的特征。这可能是阳虚体质怕冷和阴虚体质怕热的代偿性基因表达的佐证。但这两种体质均存在免疫缺陷,有患免疫缺陷性疾病和肿瘤癌症的可能。但其作用机制复杂,需要进一步研究相关作用机制。
4.阴虚体质和阳虚体质基因表达特征及差异研究,是对体质辨识法进行宏观与微观相结合的现代诠释,为形成基于体质辨识的中医健康状态识别体系提供了分子生物学依据。
Background and Objective:
Constitution identification method, which deeply reflects the stable state of the human body, is an effective way to manage health problems with Chinese characteristics. Genomics research based on constitution identification is a way to obtain the microcosmic information of health. By using genome-wide expression analysis of molecular biology of constitution characteristics we can preliminary grasp the microscopic features of biological information concerning with health.We have tried to select two typical constitution samples--yang-deficiency constitution and yin-deficiency constitution for analysis, to reveal their biological characteristics, and help microscopic identification research on health conditions, and provide the basis in identification of different constitutions, Based on the research on the microscopic biological health information,our aim is to provide the molecular basis for management of pat ient s with the yin-deficiency constitution and yang-deficiency constitution, and prevent relevant diseases. After that we may be able to do something for the health management system.
Methods:
According to the criterion of the yang-deficiency,yin-deficiency and normal constitutions, which were confirmed by the four examinat ions used by TCM doctor,12patients with the yin-deficiency constitution,12patients with of the yang-deficiency constitution and8patients with the normal constitution were enrolled in the study. Their peripheral blood sampling was collected, of the peripheral blood mononuclear cells were separated within2hours,and total RNA in white blood cells were extracted. Using an Affymetrix3'IVT expression profile chip, total DNA was synthesize and labeled.Chip synthesis and fluorescence labeling, and scanning were done. The peripheral blood of the gene expression spectrum of the subjects with yin-deficiency,yYang-deficiency and normal constitutionswas obtained. To investigate the relationship between the constitution and classification of gene by gene clustering and classifier study, and explore the application of the genetic classifier in constitution identification. At the same time, the biological function of varied expressiongenes and pathways of yin-deficiency and yang-deficiency constitution were evaluated. The purpose was to explore its molecular biological basis in healthy condition.
Results:
1. All the genes of32samples can be gathered into three categories, and this was very conform to the real Chinese medicine constitution classification.
2. Through genetic classifier which can distinguish three types of constitution on228probe, and the four parameters of sensitivity, specificity, positive predictive value,negative predictive were significant.
3. When the q-value (%)<5%and the Fold Change>2or<1/2, there is232differentially expressed genes,223were up-regulated and9down-regulated. Different genes gathed to35significant different pathways:mainly related to autoimmune disease related pathways, sugar metabolic pathways, lipid metabolic pathways, cancers pathways. Compared with normal constitution group, Yang-deficiency constitution and the yin-deficiency constitution group both have common60different expressed genes, in which there are three opposite expressed genes, nameed FFAR2, GPR109B, NAMPT. Comparison ofyin-deficiencyconstitution, Yang-deficiency constitution is raised, FFAR2(221345_at) Fold Change≈6.7796, GPR109B (205220_at) Fold Change≈7.6858, NAMPT (217739-s_at) Fold Change=6.1773(P<0.05)
4. When the q-value (%)<5%and the Fold Change>2or<1/2, there is155differentially expressed genes between yin-deficiency constitution group and normal constitution group in gene expression feature maps,92were up-regulated and63down-regulated; there is203differentially expressed genes in gene expression feature maps between yang-deficiency constitution group and normal constitution group,with51were up-regulated and152down-regulated. differentially expressed genes of yin-deficiency constitution mainly involved in the biological processes are:immune response, inflammatory response, apoptosis, DNA methylation, regulation of insulin secretion, calcium-mediated signaling, glucose metabolism, regulation of lipid catabolism, response to ATP, etc. Different genes gathed to44significant different pathways, Tcell receptor signaling pathway, Toll-like receptor signaling pathway, B cell receptor signaling pathway, Jak-STAT signal ing pathway, MAPK signal ing pathway, Systemic lupus erythematosus, Autoimmune thyroid disease, Type Ⅱ diabetes mellitus, Alzheimer's disease and many cancer parthways. Differentially expressed genes Yang-deficiency constitution of mainly involved in the biological processes are:immune response, inflammatory response, signal transduction, carbohydrate metabolism, regulation of insulin secretion, glycerol metabolism, fat cell differentiation, starch catabolism, response to ATP, oxidation reduction, etc.Different genes gathed to35significant different pathways:MAPK signaling pathway, Antigen processing and presentation, Toll-like receptor signaling pathway, T cell receptor signaling pathway, Wnt signal ing pathway、Peptidoglycan biosynthesis, B cell receptor signaling pathway, Systemic lupus erythematosus, Asthma, Autoimmune thyroid disease, Epithelial cell signaling in Helicobacter pylori infection,Alzheimer's disease,Type I diabetes mellitus, BIadder cancer, and Colorectal cancer.
Conclus ions:
1.Yin-deficiency constitution, yang-deficiency constitution and normal constitution all have their feature gene expression spectrum.
2. Chinese medicine constitution classification is very conform to the genetic classifier result. Through preliminary genetic classifier can distinguish yin-deficiency constitution, yang-deficiency constitution and normal constitution, and it provides methodology basis for health state of microscopic identification research.
3. Yin-deficiency and yang-deficiency constitution were significantly different on the gene expression. Mainly reflects that there was a big difference between the Yang-deficiency constitution and yin-deficiencyconstitution in immune function, energy metabolism, lipid metabolism, glucose metabolism, nervous system, and in tumor cancer disease, etc. Found some glucolipid metabolism related genes, expression of Yang-deficiency constitution andyin-deficiency constitution were contrary or is greatly different, Yang-deficiency constitution is far higher than that of yin-deficiency constitution. Research results show that the Yang-deficiency constitution and yin-deficiency constitution constitution was reverse differences on glucolipid metabolism, which conform to the opposite metabolism of body heat characteristics of the yang-deficiency constitution and yin-deficiency constitution. This may be an evidence that Yang-deficiency constitution feel cold and yin-deficiency constitution feel hot.But both kinds of constitution has an immune deficiency, have immune deficiency disease and tumor cancer. But its mechanism is complicated, related mechanism needs further research.
4. By studying the characteristics and differences between yin-deficiency constitution and yang-deficiency constitution in peripheral blood gene expression, This is the combination of macro and micro modern interpretation to the constitution identification method, and it provides a molecular biological basis for forming TCM health status recognition system which based on the constitution identification.
深化反映人体相对稳定状态的体质辨识法是建立中国特色健康管理的有效方法。基于体质辨识基因组学研究是获取健康状态微观信息的途径之一。利用全基因组表达谱分析体质对应的分子生物学特征,可以初步把握健康状态的微观生物信息特征。拟选取具有代表性的阳虚体质、阴虚体质两种体质进行分析,有助于揭示这两种体质的生物学特征,有助于健康状态微观辨识研究,同时为区分不同体质的人群提供依据。通过对健康状态微观生物信息的研究,最终为调理阴虚、阳虚体质提供分子生物学依据,为预防其相关性疾病的发生奠定基础,实现有效的全面的全民健康管理体系做出贡献。
方法：
按照阳虚、阴虚体质和平和体质判定标准,并由中医师四诊确认符合,最后筛选出12例阴虚质、12例阳虚体质和8例平和质。取上述受试者的外周血,2小时内分离外周血单个核细胞,提取白细胞中的总RNA.采用Affymetrix3'IVT表达谱芯片,开展cDNA的合成与标记、芯片杂交、芯片的荧光标记与扫描,获取阴虚质、阳虚质、平和质的外周血基因表达谱。进行基因聚类和基因分类器研究,探讨体质分类情况与基因聚类的关系,并探索基因分类器在体质辨识中的应用方法。同时对阴虚体质与阳虚体质的特征和差异表达基因及路通的生物学功能进行评价,探析其健康状态的分子生物学基础。
结果：
1.全部32个样本的所有基因可聚集为三类,这与样本的真实体质分类标签相符合。
2.通过基因分类器找到可区分三种体质的228个探针,并且基因分类器的敏感性、特异性、阳性预测值、阴性预测值四个参数均有意义。
3.当q-value(%)<5%并且Fold Change>2或Fold Change<1/2时,阳虚体质与阴虚体质的基因表达谱获得共232条差异表达基因,其中有223个基因表达水平上调的,有9个基因表达水平下调的。阳虚体质与阴虚体质的差异基因富集到35条差异显著的信号转导通路,主要涉及免疫功能、能量代谢、脂代谢、糖代谢、神经系统以及肿瘤癌症疾病等信号通路。同时阴虚质、阳虚质与平和质对照组比较,二者共同的差异表达基因有60条,其中表达完全相反的有3条,即FFAR2.GPR109B.NAMPT.与阴虚体质相比,阳虚体质表达远远上调,FFAR2(221345_at)Fold Change≈6.7796;GPR109B(205220_at) Fold Change≈7.6858；NAMPT(217739_s_at)Fold Change≈6.1773(P<0.05)。
4.当q-value(%)<5%并且Fold Change>2或者Fold Change<1/2时,阴虚体质组和平和体质对照组间基因表达图谱获得共155条差异表达基因,其中有92条表达水平上调,有63条表达水平下调；阳虚体质组和平和体质对照组间的基因表达图谱获得共203条差异表达基因,其中有51条表达水平上调,有152条基因表达水平下调。阴虚体质的差异表达的基因主要参与免疫应答、炎症反应、细胞凋亡、DNA甲基化、胰岛素分泌调节、钙介导的信号转导、糖代谢、脂质分解代谢、三磷酸腺苷反应等生物过程。差异基因富集到T细胞受体的信号通路、Toll-like受体信号通路、B细胞受体信号通路、Jak-STAT(?)言号通路、丝裂酶原活化蛋白激酶信号转导通路、系统性红斑狼疮、自身免疫性甲状腺疾病、Ⅱ型糖尿病、早老性痴呆及多条癌症等44条信号转导通路。阳虚体质的差异表达基因主要参与免疫应答、炎症反应、信号转导、碳水化合物代谢、调控胰岛素分泌、甘油代谢、脂肪细胞分化、淀粉分解代谢、三磷酸腺苷反应等生物过程。差异基因富集到丝裂酶原活化蛋白激酶信号转导通路、抗原加工提呈通路、tOll样受体信号通路、T细胞受体的信号通路、Wnt信号通路、肽聚糖生物合成、B细胞受体的信号通路、系统性红斑狼疮、哮喘、自身免疫性甲状腺疾病、上皮细胞幽门螺杆菌感染、早老性痴呆、I型糖尿病、膀胱癌、结肠直肠癌等30条信号转导通路。
结论：
1.阴虚体质、阳虚体质和平和体质具有各自体质的基因表达谱特征。
2.中医体质分类与基因分类结果相符合。通过基因分类器方面可以初步区分阴虚体质、阳虚体质和平和体质,同时为健康状态微观辨识研究提供方法学依据。
3.阳虚体质和阴虚体质存在基因表达图谱显著差异。差异基因主要反映阳虚体质与阴虚体质在免疫功能、能量代谢、脂代谢、糖代谢、神经系统以及肿瘤癌症疾病等方面存在很大差别。研究结果表明阳虚体质与阴虚体质在糖脂代谢上存在反向差异,这符合阳虚体质和阴虚体质热量代谢相反的特征。这可能是阳虚体质怕冷和阴虚体质怕热的代偿性基因表达的佐证。但这两种体质均存在免疫缺陷,有患免疫缺陷性疾病和肿瘤癌症的可能。但其作用机制复杂,需要进一步研究相关作用机制。
4.阴虚体质和阳虚体质基因表达特征及差异研究,是对体质辨识法进行宏观与微观相结合的现代诠释,为形成基于体质辨识的中医健康状态识别体系提供了分子生物学依据。
Background and Objective:
Constitution identification method, which deeply reflects the stable state of the human body, is an effective way to manage health problems with Chinese characteristics. Genomics research based on constitution identification is a way to obtain the microcosmic information of health. By using genome-wide expression analysis of molecular biology of constitution characteristics we can preliminary grasp the microscopic features of biological information concerning with health.We have tried to select two typical constitution samples--yang-deficiency constitution and yin-deficiency constitution for analysis, to reveal their biological characteristics, and help microscopic identification research on health conditions, and provide the basis in identification of different constitutions, Based on the research on the microscopic biological health information,our aim is to provide the molecular basis for management of pat ient s with the yin-deficiency constitution and yang-deficiency constitution, and prevent relevant diseases. After that we may be able to do something for the health management system.
Methods:
According to the criterion of the yang-deficiency,yin-deficiency and normal constitutions, which were confirmed by the four examinat ions used by TCM doctor,12patients with the yin-deficiency constitution,12patients with of the yang-deficiency constitution and8patients with the normal constitution were enrolled in the study. Their peripheral blood sampling was collected, of the peripheral blood mononuclear cells were separated within2hours,and total RNA in white blood cells were extracted. Using an Affymetrix3'IVT expression profile chip, total DNA was synthesize and labeled.Chip synthesis and fluorescence labeling, and scanning were done. The peripheral blood of the gene expression spectrum of the subjects with yin-deficiency,yYang-deficiency and normal constitutionswas obtained. To investigate the relationship between the constitution and classification of gene by gene clustering and classifier study, and explore the application of the genetic classifier in constitution identification. At the same time, the biological function of varied expressiongenes and pathways of yin-deficiency and yang-deficiency constitution were evaluated. The purpose was to explore its molecular biological basis in healthy condition.
Results:
1. All the genes of32samples can be gathered into three categories, and this was very conform to the real Chinese medicine constitution classification.
2. Through genetic classifier which can distinguish three types of constitution on228probe, and the four parameters of sensitivity, specificity, positive predictive value,negative predictive were significant.
3. When the q-value (%)<5%and the Fold Change>2or<1/2, there is232differentially expressed genes,223were up-regulated and9down-regulated. Different genes gathed to35significant different pathways:mainly related to autoimmune disease related pathways, sugar metabolic pathways, lipid metabolic pathways, cancers pathways. Compared with normal constitution group, Yang-deficiency constitution and the yin-deficiency constitution group both have common60different expressed genes, in which there are three opposite expressed genes, nameed FFAR2, GPR109B, NAMPT. Comparison ofyin-deficiencyconstitution, Yang-deficiency constitution is raised, FFAR2(221345_at) Fold Change≈6.7796, GPR109B (205220_at) Fold Change≈7.6858, NAMPT (217739-s_at) Fold Change=6.1773(P<0.05)
4. When the q-value (%)<5%and the Fold Change>2or<1/2, there is155differentially expressed genes between yin-deficiency constitution group and normal constitution group in gene expression feature maps,92were up-regulated and63down-regulated; there is203differentially expressed genes in gene expression feature maps between yang-deficiency constitution group and normal constitution group,with51were up-regulated and152down-regulated. differentially expressed genes of yin-deficiency constitution mainly involved in the biological processes are:immune response, inflammatory response, apoptosis, DNA methylation, regulation of insulin secretion, calcium-mediated signaling, glucose metabolism, regulation of lipid catabolism, response to ATP, etc. Different genes gathed to44significant different pathways, Tcell receptor signaling pathway, Toll-like receptor signaling pathway, B cell receptor signaling pathway, Jak-STAT signal ing pathway, MAPK signal ing pathway, Systemic lupus erythematosus, Autoimmune thyroid disease, Type Ⅱ diabetes mellitus, Alzheimer's disease and many cancer parthways. Differentially expressed genes Yang-deficiency constitution of mainly involved in the biological processes are:immune response, inflammatory response, signal transduction, carbohydrate metabolism, regulation of insulin secretion, glycerol metabolism, fat cell differentiation, starch catabolism, response to ATP, oxidation reduction, etc.Different genes gathed to35significant different pathways:MAPK signaling pathway, Antigen processing and presentation, Toll-like receptor signaling pathway, T cell receptor signaling pathway, Wnt signal ing pathway、Peptidoglycan biosynthesis, B cell receptor signaling pathway, Systemic lupus erythematosus, Asthma, Autoimmune thyroid disease, Epithelial cell signaling in Helicobacter pylori infection,Alzheimer's disease,Type I diabetes mellitus, BIadder cancer, and Colorectal cancer.
Conclus ions:
1.Yin-deficiency constitution, yang-deficiency constitution and normal constitution all have their feature gene expression spectrum.
2. Chinese medicine constitution classification is very conform to the genetic classifier result. Through preliminary genetic classifier can distinguish yin-deficiency constitution, yang-deficiency constitution and normal constitution, and it provides methodology basis for health state of microscopic identification research.
3. Yin-deficiency and yang-deficiency constitution were significantly different on the gene expression. Mainly reflects that there was a big difference between the Yang-deficiency constitution and yin-deficiencyconstitution in immune function, energy metabolism, lipid metabolism, glucose metabolism, nervous system, and in tumor cancer disease, etc. Found some glucolipid metabolism related genes, expression of Yang-deficiency constitution andyin-deficiency constitution were contrary or is greatly different, Yang-deficiency constitution is far higher than that of yin-deficiency constitution. Research results show that the Yang-deficiency constitution and yin-deficiency constitution constitution was reverse differences on glucolipid metabolism, which conform to the opposite metabolism of body heat characteristics of the yang-deficiency constitution and yin-deficiency constitution. This may be an evidence that Yang-deficiency constitution feel cold and yin-deficiency constitution feel hot.But both kinds of constitution has an immune deficiency, have immune deficiency disease and tumor cancer. But its mechanism is complicated, related mechanism needs further research.
4. By studying the characteristics and differences between yin-deficiency constitution and yang-deficiency constitution in peripheral blood gene expression, This is the combination of macro and micro modern interpretation to the constitution identification method, and it provides a molecular biological basis for forming TCM health status recognition system which based on the constitution identification.
引文
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