發(fā)布時間：2018-03-14 08:52

  本文選題：代謝組學(xué)　切入點：呼吸道合胞病毒　出處：《南京中醫(yī)藥大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：背景：肺炎是嬰幼兒的常見病,是嬰幼兒死亡的重要原因,被世界衛(wèi)生組織(World Health Organization, WHO)列為全球三種重要兒科疾病之一,我國的病毒性肺炎隨著社會發(fā)展表現(xiàn)出明顯的上升趨勢。小兒病毒性肺炎病原體常見：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。本病在中醫(yī)學(xué)屬于“肺炎喘嗽”范疇,而痰熱閉肺證是本病發(fā)病機(jī)率最高、最重要的證候。中醫(yī)的證候與現(xiàn)代醫(yī)學(xué)之間目前尚無直接和必然的聯(lián)系,很難用一個或者幾個指標(biāo)來證實某個中醫(yī)證候�？茖W(xué)而全面地探索中醫(yī)證候的物質(zhì)基礎(chǔ)是中醫(yī)現(xiàn)代化進(jìn)程中急需解決的關(guān)鍵問題。相關(guān)研究尚無明確證據(jù)證實西藥對于小兒病毒性肺炎的針對性治療作用,目前僅有“利巴韋林”被美國兒科協(xié)會推薦為“也許可以使用”。相比較于西醫(yī)治療小兒病毒性肺炎的明顯不足,中醫(yī)中藥具有明顯的優(yōu)勢,并且已經(jīng)得到諸多臨床研究和實驗研究的證實。南京中醫(yī)藥大學(xué)汪受傳教授領(lǐng)銜本課題組,致力于中醫(yī)藥治療小兒病毒性肺炎的相關(guān)研究20年,不但確認(rèn)了本病的證候分型、治則治法,制訂了診療指南,還研制了具有宣肺開閉、清熱解毒、化痰止咳功效的金欣口服液。此外還有諸如炎琥寧、清開靈、喜炎平等許多中藥注射劑和品類繁多的中藥口服劑在小兒病毒性肺炎的治療中發(fā)揮著重要作用。代謝組學(xué)(Metabonomics)作為系統(tǒng)生物學(xué)的一門新興學(xué)科,重點關(guān)注生物體內(nèi)源性代謝物的種類、數(shù)量和變化規(guī)律。生物體的內(nèi)源性代謝物是基因和蛋白活動真實、靈敏的最終產(chǎn)物,代謝組學(xué)不著眼于某個或者某類物質(zhì),它的研究對象是生物體的全部內(nèi)源性代謝物。所以,應(yīng)用代謝組學(xué)進(jìn)行研究可以避免傳統(tǒng)研究方法中過分關(guān)注某一個或者幾個指標(biāo)變化的不足。代謝組學(xué)在方法學(xué)上擁有整體性、動態(tài)性和綜合性的特點。這樣的特點跟中醫(yī)理論最重要和最基礎(chǔ)的“整體觀”和“動態(tài)觀”不謀而合,在哲學(xué)思辨層次上,現(xiàn)代科技前沿的代謝組學(xué)與古老的中醫(yī)學(xué)擁有了可以溝通的語言。因此本課題組采用代謝組學(xué)方法考察上述4種病毒導(dǎo)致的小兒病毒性肺炎痰熱閉肺證患兒的血漿樣本中代謝物組的變化及其規(guī)律,尋找痰熱閉肺證的標(biāo)志性代謝物組,嘗試闡明小兒病毒性肺炎痰熱閉肺證的科學(xué)實質(zhì)。目的：以代謝組學(xué)為技術(shù)平臺,分別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)感染所致小兒病毒性肺炎的痰熱閉肺證的血漿樣本,探索代謝物組、代謝通路的變化及其規(guī)律,探討小兒病毒性肺炎痰熱閉肺證的證候?qū)嵸|(zhì)。方法：小兒病毒性肺炎痰熱閉肺證代謝組學(xué)研究的臨床實驗：依據(jù)病毒性肺炎和痰熱閉肺證的相關(guān)中醫(yī)、西醫(yī)診斷標(biāo)準(zhǔn),排除不適宜樣本,收集每種病毒肺炎患兒的血漿樣本30例左右設(shè)為疾病組,同時收集健康兒童的血漿樣本30例,設(shè)為正常組。實際入選樣本RSV組35例、ADV組31例、IFV組11例、PIV組32例、正常組30例。采用超高效液相-二維線性離子阱質(zhì)譜聯(lián)用儀(UPLC-LTQ/Orbitrap-MS)分別檢測4個肺炎組樣本及正常組樣本,采集小兒病毒性肺炎痰熱閉肺證的證候代謝譜及正常兒童的代謝譜,利用XCMS Online云平臺采用主成份分析(Principal Component Analysis, PCA)、正交偏最小二乘法(Orthogonal Partial Least Squares Disceiminant Analysis, OPLS-DA)、非線性多維標(biāo)度分析(Non-Metric Multidimensional Scaling, NMMDS)等分析方法對所得矩陣數(shù)據(jù)進(jìn)行統(tǒng)計分析,篩選每個病毒性肺炎組相對與正常組的內(nèi)源性代謝物組作為潛在證候?qū)W生物標(biāo)記物組,采用Mummichog等分析算法連接在線METLIN數(shù)據(jù)庫、HMDB數(shù)據(jù)庫、UMDB數(shù)據(jù)庫、LIPID MAPs數(shù)據(jù)庫等數(shù)據(jù)庫分析每組病毒性肺炎的代謝通路和代謝模型,探索不同病毒所致小兒病毒性肺炎痰熱閉肺證患兒與正常兒童之間典型的差異性代謝物。得出以上研究結(jié)果之后本研究不設(shè)動物實驗及藥物治療對比實驗,而是在真實世界研究范式下橫向綜合全部4組研究結(jié)果,從中找出不同病毒導(dǎo)致同一痰熱閉肺證的共同代遂：物組,分析全部痰熱閉肺證樣本受到4種不同病毒組影響而代謝物變化趨勢完全一致的核心代謝物組,形成小兒病毒性肺炎痰熱閉肺證的代謝物密碼表。結(jié)果：1.研究發(fā)現(xiàn)ADV肺炎痰熱閉肺證患兒的血漿主要以7alpha-Hydroxycholest-4-en-3-one(膽汁酸前體)、二氫睪酮、脫氫皮質(zhì)甾酮、二氫葉酸、脂肪酸、維生素C等代謝紊亂為主,與正常兒童的血漿樣本可以完全區(qū)分。2.研究發(fā)現(xiàn)RSV肺炎痰熱閉肺證患兒的血漿主要以色氨酸代謝、維生素C和aldarate代謝、卟啉代謝、酪氨酸代謝、天門冬氨酸和天冬酰胺代謝、賴氨酸代謝、嘌呤代謝、飽和脂肪酸的β-氧化等代謝紊亂為主,與正常兒童的血漿樣本可以完全區(qū)分。3.研究發(fā)現(xiàn)IFV肺炎痰熱閉肺證患兒的血漿主要以肉堿穿梭、維生素E、唾液酸、天門冬氨酸和天冬酰胺代謝,谷氨酸代謝,精氨酸和脯氨酸代謝,甘氨酸,絲氨酸,丙氨酸,蘇氨酸代謝,維生素B9(葉酸)代謝,生物喋呤代謝,脂肪酸代謝,酪氨酸代謝,從頭脂肪酸生物合成,丙氨酸和天門冬氨酸代謝,維生素A(視黃醇)代謝等代謝紊亂為主,與正常兒童的血漿樣本可以完全區(qū)分。4.研究發(fā)現(xiàn)PIV肺炎痰熱閉肺證患兒的血漿主要以肉堿穿梭、生物喋呤代謝、維生素E代謝、維生素D3代謝、強(qiáng)啡肽代謝、維生素B9(葉酸)代謝、角鯊烯和膽固醇的生物合成、賴氨酸代謝、膽汁酸生物合成、鞘糖脂代謝、丁酸代謝等,涉及到了肉堿、維生素、激素、氨基酸、膽固醇等等代謝紊亂為主,與正常兒童的血漿樣本可以完全區(qū)分。5.對ADV、RSV、IFV、 PIV四種病毒所致病毒性肺炎痰熱閉肺證進(jìn)行兩兩對比分析,闡明四種病毒兩兩對比共計6組典型的差異性代謝通路。6.以真實世界研究范式考察4種病毒性肺炎與正常兒童血漿代謝物組的不同,找出其上層血漿313種核心代謝物和下層血漿128種核心代謝物,并從中篩選出來四種病毒影響趨勢完全一致的標(biāo)志性核心物質(zhì),其中上調(diào)71種、下調(diào)349種、共計420種代謝物；標(biāo)明其趨勢及其調(diào)整倍數(shù),形成小兒病毒性肺炎痰熱閉肺證的物質(zhì)基礎(chǔ)密碼表。結(jié)論：1.采用XCMS Online平臺以“云存儲”與“云計算”技術(shù),用代謝組學(xué)分析不同病毒導(dǎo)致的肺炎痰熱閉肺證患兒血漿的代謝物組變化,用大數(shù)據(jù)研究方法闡釋中醫(yī)證候的物質(zhì)基礎(chǔ)切實可行,結(jié)果可靠。2.從ADV、RSV、IFV、PIV等四種病毒性肺炎痰熱閉肺證入手,以UPLC-LTQ/Orbitrap-MS實驗技術(shù)平臺從代謝組學(xué)分析不同病毒導(dǎo)致的肺炎痰熱閉肺證的代謝紊亂表現(xiàn),可以闡明并區(qū)分ADV、RSV、IFV、PIV肺炎痰熱閉肺證與健康兒童的不同代謝模式。3.病毒性肺炎對小兒影響主要造成內(nèi)源性代謝物含量的降低,而調(diào)高的代謝物整體上占較低比例。從代謝組學(xué)角度說明了小兒體質(zhì)應(yīng)當(dāng)描述為“稚陰稚陽”而非“陽常有余”。4.通過橫向分析4種病毒所致小兒病毒性肺炎痰熱閉肺證血漿代謝模式紊亂結(jié)果,得到420種調(diào)整趨勢完全一致的代謝物,證明確實存在超越病毒種類的中醫(yī)特定證候的標(biāo)志性代謝物組。
[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.

【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R272
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本文編號：1610492