基于UPLC-MS技术的小儿病毒性肺炎痰热闭肺证代谢模式研究

发布时间：2018-03-14 08:52

  本文选题：代谢组学　切入点：呼吸道合胞病毒　出处：《南京中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：背景：肺炎是婴幼儿的常见病,是婴幼儿死亡的重要原因,被世界卫生组织(World Health Organization, WHO)列为全球三种重要儿科疾病之一,我国的病毒性肺炎随着社会发展表现出明显的上升趋势。小儿病毒性肺炎病原体常见：呼吸道合胞病毒(Respiratory syncytial virus, RSV)、腺病毒(Adenovirus, ADV)、流感病毒(Influenza virus, IFV)、副流感病毒(Parainfluenza virus, PIV)等病毒。本病在中医学属于“肺炎喘嗽”范畴,而痰热闭肺证是本病发病机率最高、最重要的证候。中医的证候与现代医学之间目前尚无直接和必然的联系,很难用一个或者几个指标来证实某个中医证候。科学而全面地探索中医证候的物质基础是中医现代化进程中急需解决的关键问题。相关研究尚无明确证据证实西药对于小儿病毒性肺炎的针对性治疗作用,目前仅有“利巴韦林”被美国儿科协会推荐为“也许可以使用”。相比较于西医治疗小儿病毒性肺炎的明显不足,中医中药具有明显的优势,并且已经得到诸多临床研究和实验研究的证实。南京中医药大学汪受传教授领衔本课题组,致力于中医药治疗小儿病毒性肺炎的相关研究20年,不但确认了本病的证候分型、治则治法,制订了诊疗指南,还研制了具有宣肺开闭、清热解毒、化痰止咳功效的金欣口服液。此外还有诸如炎琥宁、清开灵、喜炎平等许多中药注射剂和品类繁多的中药口服剂在小儿病毒性肺炎的治疗中发挥着重要作用。代谢组学(Metabonomics)作为系统生物学的一门新兴学科,重点关注生物体内源性代谢物的种类、数量和变化规律。生物体的内源性代谢物是基因和蛋白活动真实、灵敏的最终产物,代谢组学不着眼于某个或者某类物质,它的研究对象是生物体的全部内源性代谢物。所以,应用代谢组学进行研究可以避免传统研究方法中过分关注某一个或者几个指标变化的不足。代谢组学在方法学上拥有整体性、动态性和综合性的特点。这样的特点跟中医理论最重要和最基础的“整体观”和“动态观”不谋而合,在哲学思辨层次上,现代科技前沿的代谢组学与古老的中医学拥有了可以沟通的语言。因此本课题组采用代谢组学方法考察上述4种病毒导致的小儿病毒性肺炎痰热闭肺证患儿的血浆样本中代谢物组的变化及其规律,寻找痰热闭肺证的标志性代谢物组,尝试阐明小儿病毒性肺炎痰热闭肺证的科学实质。目的：以代谢组学为技术平台,分别研究呼吸道合胞病毒(RSV)、腺病毒(ADV)、流感病毒(IFV)、副流感病毒(PIV)感染所致小儿病毒性肺炎的痰热闭肺证的血浆样本,探索代谢物组、代谢通路的变化及其规律,探讨小儿病毒性肺炎痰热闭肺证的证候实质。方法：小儿病毒性肺炎痰热闭肺证代谢组学研究的临床实验：依据病毒性肺炎和痰热闭肺证的相关中医、西医诊断标准,排除不适宜样本,收集每种病毒肺炎患儿的血浆样本30例左右设为疾病组,同时收集健康儿童的血浆样本30例,设为正常组。实际入选样本RSV组35例、ADV组31例、IFV组11例、PIV组32例、正常组30例。采用超高效液相-二维线性离子阱质谱联用仪(UPLC-LTQ/Orbitrap-MS)分别检测4个肺炎组样本及正常组样本,采集小儿病毒性肺炎痰热闭肺证的证候代谢谱及正常儿童的代谢谱,利用XCMS Online云平台采用主成份分析(Principal Component Analysis, PCA)、正交偏最小二乘法(Orthogonal Partial Least Squares Disceiminant Analysis, OPLS-DA)、非线性多维标度分析(Non-Metric Multidimensional Scaling, NMMDS)等分析方法对所得矩阵数据进行统计分析,筛选每个病毒性肺炎组相对与正常组的内源性代谢物组作为潜在证候学生物标记物组,采用Mummichog等分析算法连接在线METLIN数据库、HMDB数据库、UMDB数据库、LIPID MAPs数据库等数据库分析每组病毒性肺炎的代谢通路和代谢模型,探索不同病毒所致小儿病毒性肺炎痰热闭肺证患儿与正常儿童之间典型的差异性代谢物。得出以上研究结果之后本研究不设动物实验及药物治疗对比实验,而是在真实世界研究范式下横向综合全部4组研究结果,从中找出不同病毒导致同一痰热闭肺证的共同代遂：物组,分析全部痰热闭肺证样本受到4种不同病毒组影响而代谢物变化趋势完全一致的核心代谢物组,形成小儿病毒性肺炎痰热闭肺证的代谢物密码表。结果：1.研究发现ADV肺炎痰热闭肺证患儿的血浆主要以7alpha-Hydroxycholest-4-en-3-one(胆汁酸前体)、二氢睾酮、脱氢皮质甾酮、二氢叶酸、脂肪酸、维生素C等代谢紊乱为主,与正常儿童的血浆样本可以完全区分。2.研究发现RSV肺炎痰热闭肺证患儿的血浆主要以色氨酸代谢、维生素C和aldarate代谢、卟啉代谢、酪氨酸代谢、天门冬氨酸和天冬酰胺代谢、赖氨酸代谢、嘌呤代谢、饱和脂肪酸的β-氧化等代谢紊乱为主,与正常儿童的血浆样本可以完全区分。3.研究发现IFV肺炎痰热闭肺证患儿的血浆主要以肉碱穿梭、维生素E、唾液酸、天门冬氨酸和天冬酰胺代谢,谷氨酸代谢,精氨酸和脯氨酸代谢,甘氨酸,丝氨酸,丙氨酸,苏氨酸代谢,维生素B9(叶酸)代谢,生物喋呤代谢,脂肪酸代谢,酪氨酸代谢,从头脂肪酸生物合成,丙氨酸和天门冬氨酸代谢,维生素A(视黄醇)代谢等代谢紊乱为主,与正常儿童的血浆样本可以完全区分。4.研究发现PIV肺炎痰热闭肺证患儿的血浆主要以肉碱穿梭、生物喋呤代谢、维生素E代谢、维生素D3代谢、强啡肽代谢、维生素B9(叶酸)代谢、角鲨烯和胆固醇的生物合成、赖氨酸代谢、胆汁酸生物合成、鞘糖脂代谢、丁酸代谢等,涉及到了肉碱、维生素、激素、氨基酸、胆固醇等等代谢紊乱为主,与正常儿童的血浆样本可以完全区分。5.对ADV、RSV、IFV、 PIV四种病毒所致病毒性肺炎痰热闭肺证进行两两对比分析,阐明四种病毒两两对比共计6组典型的差异性代谢通路。6.以真实世界研究范式考察4种病毒性肺炎与正常儿童血浆代谢物组的不同,找出其上层血浆313种核心代谢物和下层血浆128种核心代谢物,并从中筛选出来四种病毒影响趋势完全一致的标志性核心物质,其中上调71种、下调349种、共计420种代谢物；标明其趋势及其调整倍数,形成小儿病毒性肺炎痰热闭肺证的物质基础密码表。结论：1.采用XCMS Online平台以“云存储”与“云计算”技术,用代谢组学分析不同病毒导致的肺炎痰热闭肺证患儿血浆的代谢物组变化,用大数据研究方法阐释中医证候的物质基础切实可行,结果可靠。2.从ADV、RSV、IFV、PIV等四种病毒性肺炎痰热闭肺证入手,以UPLC-LTQ/Orbitrap-MS实验技术平台从代谢组学分析不同病毒导致的肺炎痰热闭肺证的代谢紊乱表现,可以阐明并区分ADV、RSV、IFV、PIV肺炎痰热闭肺证与健康儿童的不同代谢模式。3.病毒性肺炎对小儿影响主要造成内源性代谢物含量的降低,而调高的代谢物整体上占较低比例。从代谢组学角度说明了小儿体质应当描述为“稚阴稚阳”而非“阳常有余”。4.通过横向分析4种病毒所致小儿病毒性肺炎痰热闭肺证血浆代谢模式紊乱结果,得到420种调整趋势完全一致的代谢物,证明确实存在超越病毒种类的中医特定证候的标志性代谢物组。
[Abstract]:Background: pneumonia is a common disease in infants, is an important cause of infant death, by the WHO (World Health Organization, WHO) as one of the world's three major pediatric diseases, viral pneumonia in China with the development of the society showed a clear upward trend. The common pathogen of viral pneumonia in children: respiratory syncytial virus (Respiratory syncytial virus, RSV), adenovirus (Adenovirus, ADV), influenza virus (Influenza, virus, IFV), parainfluenza virus (Parainfluenza virus, PIV) virus. The disease belongs to "pneumonia" category in TCM, and the syndrome of phlegm heat in the lung is the highest incidence of disease, syndromes of the important. There is no direct and inevitable connection between TCM syndromes and modern medicine, it is difficult to use one or several indicators to confirm a TCM syndrome. Scientific and comprehensive exploration of the material basis of TCM Syndrome The foundation is the key problem needed to solve in the process of modernization of traditional Chinese medicine. There is no clear evidence of Western medicine for viral pneumonia in children for treatment, is currently the only "ribavirin is the American Pediatric Association recommended as the" may use ". Obviously insufficient compared to western medicine in the treatment of viral pneumonia in children, with traditional Chinese medicine the obvious advantage, and has been confirmed in many clinical and experimental studies. The Nanjing University of Chinese Medicine Wang led the research group by the missionary award, 20 years of research related to Chinese medicine to treat viral pneumonia, not only confirmed this disease syndrome type, therapeutic principle, formulate guidelines, as well as the development of with Xuanfei opening and closing, detoxification, Jinxin oral liquid Huatanzhike effect. In addition to such Yanhuning, Qingkailing, like many traditional Chinese medicine injections and arthritis equality category Play an important role in the treatment of traditional Chinese medicine oral preparation of various children's virus pneumonia. Metabonomics (Metabonomics) as an emerging discipline of systems biology, focusing on the type of organism endogenous metabolites, and the number of changes of endogenous metabolites. The organism is gene and protein activity, the final product is sensitive. Metabolomics does not focus on one or a group of substances, which is the object of study organisms all endogenous metabolites. Therefore, the application of metabonomics study can avoid the traditional research methods too close enough note one or several indexes. Metabonomics in methodology with integrity, dynamic characteristics and comprehensive. The "whole" and "dynamic" view of the most important characteristics of such agree without prior without previous consultation with the Chinese medicine theory and the foundation, in the philosophical level, modern science and technology The metabolism of group learning and frontier of ancient TCM have can communicate with language. So the research group used metabolomics metabolomics samples of plasma viral pneumonia in children of phlegm and heat in the above 4 virus causes the methodology of the lung were closed in the change and law, finding landmark metabolome of phlegm heat obstructing the lung syndrome the essence of science, try to clarify the infantile viral pneumonia with syndrome of phlegm heat in the lung. Objective: using metabonomics technology platform, respectively study on respiratory syncytial virus (RSV), adenovirus (ADV), influenza virus (IFV), parainfluenza virus (PIV) plasma samples of children with viral infection caused by phlegm heat pneumonia closed the lung, explore the metabolome, changes of metabolic pathway, essence of syndromes of viral pneumonia in children of phlegm heat closed the lung. Methods: clinical and experimental lung syndrome Metabonomics Study closed viral pneumonia in children: on the basis of phlegm heat Viral pneumonia and related TCM syndrome of phlegm heat in the lung, Western diagnostic criteria, excluding the suitable sample, the plasma samples were collected for each virus pneumonia in 30 cases located around the disease group, 30 cases of plasma samples were collected at the same time of healthy children served as normal group. The sample selected 35 cases in RSV group and ADV group in 31 cases, 11 cases of IFV group, PIV group of 32 cases, 30 cases of normal group. By using ultra performance liquid chromatography two-dimensional linear ion trap mass spectrometry (UPLC-LTQ/Orbitrap-MS) were detected in 4 samples of the pneumonia group and normal group samples, and normal children metabolic syndrome acquisition of viral pneumonia in children the accumulation of phlegm heat in lung the metabolic spectrum spectrum, using the XCMS Online cloud platform by using the principal component analysis (Principal Component, Analysis, PCA), orthogonal partial least squares method (Orthogonal Partial Least Squares Disceiminant Analysis, OPLS-DA), the degree of nonlinear analysis of multidimensional scaling (Non-Metric Multidimensiona L Scaling, NMMDS) and other analysis methods for statistical analysis on the data matrix, screening each viral pneumonia group and the normal group relative to endogenous metabolomic biomarkers as potential syndromes, using the Mummichog analysis algorithm connected online METLIN database, HMDB database, UMDB database, metabolic pathways and metabolic models in LIPID MAPs database each such database analysis of viral pneumonia, explore different virus induced viral pneumonia in children of phlegm heat in lung syndrome between normal children and children with typical differences in metabolites. After draws the above research results this study does not establish a comparative experimental animal experiment and drug treatment, but in the real world study paradigm under lateral integration of all 4 groups of the results, find out the different virus causes the same syndrome of phlegm heat in the lung common generation: group, then analyze all kind of the syndrome of phlegm heat in the lung was 4 The influence of different virus groups metabolites change trend is consistent with core metabolites, the formation of infantile viral pneumonia with phlegm heat closed the lung metabolites password table. The results of 1. studies found that ADV pneumonia with syndrome of phlegm heat in the lung plasma mainly 7alpha-Hydroxycholest-4-en-3-one (bile acid precursors), two hydrogen testosterone, dehydrocorticosterone dihydrofolic acid, fatty acid, vitamin C and other metabolic disorders, and plasma samples of normal children can distinguish.2. found RSV pneumonia in children with the syndrome of phlegm heat in the lung mainly in plasma tryptophan metabolism, vitamin C and aldarate metabolism, porphyrin metabolism, tyrosine metabolism, aspartic acid and asparagine lysine metabolism. Metabolism, purine metabolism, fatty acid beta oxidation and metabolic disorders, and plasma samples of normal children can distinguish.3. found IFV pneumonia syndrome of phlegm heat in the lung in children with blood Pulp mainly carnitine shuttle, vitamin E, sialic acid, aspartic acid and asparagine metabolism, glutamate metabolism, arginine and proline metabolism, glycine, serine, alanine, threonine metabolism, vitamin B9 (folic acid) metabolism, tetrahydrobiopterin metabolism, fatty acid metabolism, tyrosine metabolism, de novo fatty acid biosynthesis, and alanine aspartic acid metabolism, vitamin A (retinol) metabolism and other metabolic disorders, and plasma samples of normal children can distinguish.4. found PIV pneumonia in children with the syndrome of phlegm heat in the lung to plasma carnitine shuttle, tetrahydrobiopterin metabolism, metabolism of vitamin E, vitamin D3 metabolism, dynorphin metabolism, vitamin B9 (folic acid) metabolism, biosynthesis of squalene and cholesterol, lysine metabolism, bile acid biosynthesis, glycosphingolipid metabolism, butyric acid metabolism, involving carnitine, vitamins, hormones, amino acids, cholesterol and so on Metabolic disorders, and plasma samples of normal children can distinguish between.5. to ADV, RSV, IFV, PIV four kinds of virus induced viral pneumonia with syndrome of phlegm heat in the lung of 22 comparative analysis, clarify the comparison of four kinds of virus 22 total differences of 6 typical metabolic pathways of.6. group on the paradigm real world study investigated 4 kinds of virus pneumonia in children with normal plasma metabolites in different groups, the upper plasma metabolites and lower plasma 313 core 128 core metabolites, and from the selected landmark core material of four kinds of virus effect of the trend of exactly the same, which increased 71, down 349, a total of 420 metabolites; indicating the trend and its adjustment the formation of multiple infantile viral pneumonia with phlegm heat closed the lung is the material basis of the password table. Conclusion: 1. using the XCMS Online platform to "cloud storage" and "cloud computing" technology, using metabonomics analysis Changes of metabolites in pneumonia with phlegm heat obstructing the lung syndrome caused by the same virus in plasma, with the material basis of big data research methods explain TCM syndrome is feasible, reliable results from.2. ADV, RSV, IFV, PIV and other four kinds of viral pneumonia with syndrome of phlegm heat in the lung with metabolic disorders in experimental technology platform UPLC-LTQ/ Orbitrap-MS from the metabonomic analysis of different viruses cause pneumonia phlegm heat closed lung syndrome, can clarify and distinguish between ADV, RSV, IFV, effects of different metabolic patterns of viral pneumonia.3. PIV pneumonia with syndrome of phlegm heat in the lung and healthy children in children is mainly caused by reducing the content of endogenous metabolites, and increased metabolites on the whole accounted for a lower proportion. The children's constitution should be described as "juvenile Yin and Yang" instead of "Yang often than".4. through the horizontal analysis of 4 kinds of virus induced viral pneumonia in children phlegm heat closed the lung from the perspective of metabolic plasma generation The results showed that there were 420 kinds of metabolites that had the same trend of adjustment.

【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R272
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本文编号：1610492