本文選題：移植供肝　切入點：代謝組學　出處：《新鄉(xiāng)醫(yī)學院》2015年碩士論文　論文類型：學位論文

【摘要】：目的本研究擬以核磁共振氫譜(1H nuclear magnetic resonance,1HNMR)代謝組學技術(shù)為基礎(chǔ),在臨床實際中建立移植肝臟保存模型,對移植肝臟低溫保存中不同時間節(jié)點的代謝組進行研究,分析在低溫保存中的代謝變化,建立以核磁共振氫譜(1Hnuclear magnetic resonance,1HNMR)代謝組學在移植供肝質(zhì)量和功能評估中新方法。方法(1)隨機選取40例低溫保存移植肝臟,供體年齡控制在50歲以內(nèi),性別隨機分配。根據(jù)離體肝臟保存的不同時間(基點Oh、4h、8h、12h)每例取保存液2ml,每個時間點10例樣本,收集到的UW液應(yīng)自門靜脈原位灌注后,至肝臟顏色變?yōu)橥咙S色,從上下腔靜脈和下下腔靜脈流出收集,收集后立即放置液氮中保存以備使用。首先對采集到樣本制備,然后使用Varian UNITY INOVA 600 MHz超導脈沖傅立葉變換核磁共振譜儀采集保存液樣本和組織提取物的核磁共振數(shù)據(jù)(NMR)。NMR圖譜是由自由感應(yīng)衰減(free induction decay,FID)信號經(jīng)超導脈沖傅立葉轉(zhuǎn)換而來,最后所有的數(shù)據(jù)輸出并保存,用于模式識別分析。把保存的數(shù)據(jù)先由Pareto標度化(Pareto scaling)和平均中心化(mean centering)進行預處理,再由 SMCA-P12.0軟件(v12.0, Umetrics, Ume,Sweden)將得到的數(shù)據(jù)采用主成分分析法(principal component analysis, PCA)分析,為強化組間差異,進一步采用偏最小二乘法判別分析(PLS-DA)或正交信號校正(OSC)分析。以得分圖(scores plot)和因子載荷圖(loadings plot)的形式表示分析結(jié)果。四組保存液樣本代謝物的歸一化積分值采用統(tǒng)計學SPSS 17.0對單因素方差分析(ANOVA),P0.05認為差異具有統(tǒng)計學意義。(2)建立以1HNMR代謝組學技術(shù)為基礎(chǔ)的肝臟保存液代謝組學平臺,探討1HNMR技術(shù)在移植肝臟保存中功能評估和質(zhì)量評估應(yīng)用的可行性,并通過UW液的在不同時期的代謝變化,同時找出引起這些差異變化的主要代謝物,作為潛在的標志物。結(jié)果1、1H-NMR譜圖中可以看出,UW保存液中棉子糖(Raffinose)、腺苷(Adenosine)、檸檬酸鹽(Citrate)、琥珀酸鹽(Succinate)、乙酸(Ethanol)、肌酸/肌酐(creatine/creatinine)、次黃苷(Inosine)、乳酸(Lactate)丙氨酸、等代謝物的含量在不同時期發(fā)生了變化,而次黃嘌呤(Allopurinol)、谷胱甘肽(Glutathione)、膽堿(Choline)、等代謝物則變化不明顯。2、對0h、4h、8h、12h混合組先進行了PCA和OPLS-DA分析。其中Oh灌注的UW液樣本有明顯的聚集情況,而4h、8h及12h明顯的與0h分開,且較為分散,從PCA載荷圖是可以看出明顯的差別的,說明隨著保存時間的延長,各組都發(fā)生了明顯的代謝變化,是但4h、8h、12h樣本之間的氫譜圖之間存在一定重疊。3、在4h與8h、4h與12h以及8h與12h之間進行了PCA和OPLS-DA模式識別分析,各組之間代謝差異明顯,特別是顯現(xiàn)在OPLS-DA模式識別分析。找出每組之間差異明顯的積分區(qū)段所對應(yīng)的代謝物質(zhì),也是影響著移植供肝代謝變化明顯的生物標志物。其中琥珀酸鹽、檸檬酸鹽、乳酸、谷氨酰胺以及次黃苷,腺嘌呤、葡萄糖的代謝是引起4h、8h、12h組與基準點差異最大的代謝物。結(jié)論1、基于1HNMR的UW液代謝組學研究方法可以通過研究正常組和保存組移植供肝UW液的小分子內(nèi)源性代謝物的變化,找到能夠?qū)嶒炛邪l(fā)現(xiàn)能夠代表移植供肝質(zhì)量用以評估的生物標志物：琥珀酸鹽、檸檬酸鹽、乳酸、谷氨酰胺以及次黃苷等,為評價移植供肝質(zhì)量評估提供新的思路和方法。2、這些標志物與糖代謝的無氧酵解,三羧酸循環(huán)及腺嘌呤代謝密切相關(guān),證實肝臟在人體的新陳代謝中重要的作用。
[Abstract]:The purpose of this study on proton magnetic resonance spectroscopy (1H nuclear magnetic resonance, 1HNMR) metabonomics technology as the foundation, the establishment of transplanted liver preservation model in clinical practice, to study the metabolism of liver transplantation group at different time nodes in low temperature preservation, analysis of metabolic changes in cryopreservation, to establish nuclear magnetic resonance spectroscopy (1Hnuclear magnetic resonance, 1HNMR) in transplanted liver function and quality assessment method of metabolic group. Methods (1) randomly selected 40 cases of cryopreservation of liver transplantation, donor age control in less than 50 years old, were randomly assigned to gender. According to the different time from liver preservation (point Oh 4h, 8h, 12h.) from each patient preservation solution 2ml, each time point 10 samples, UW liquid collected from the portal vein in situ perfusion to the liver, the color was yellow, the outflow from the inferior vena cava and inferior vena cava were collected immediately after collection, put Stored in liquid nitrogen for use. First of all the collected samples were prepared, and then use the Varian UNITY INOVA 600 MHz superconducting pulsed Fu Liye transform nuclear magnetic resonance NMR data acquisition instrument and sample preservation solution of tissue extracts (NMR) of.NMR was attenuated by free induction (free induction decay, FID) signal by superconducting pulsed Fu Liye when all the data, finally output and preserved for pattern recognition analysis. The data stored by the Pareto scale (Pareto scaling) and the average center (mean centering) were pretreated by SMCA-P12.0 software (v12.0, Umetrics, Ume, Sweden) will receive data using principal component analysis method (principal component analysis, PCA) analysis, in order to strengthen further the differences between groups, using partial least squares discriminant analysis (PLS-DA) and orthogonal signal correction (OSC) analysis. To score (scores plot) map And the factor loading graph (loadings plot) said the results were compared with SPSS. 17 of single factor variance normalized scores of four groups sample preservation liquid metabolites (ANOVA), P0.05 believes that the difference was statistically significant. (2) to establish 1HNMR metabolomics technology based metabolomics liver preservation solution to investigate the feasibility of 1HNMR Technology platform, the function in the transplanted liver preservation evaluation and quality evaluation, and through the UW fluid metabolism in different periods, and find out the main metabolites caused by these differences change, as a potential marker. The results of 1,1H-NMR spectra can be seen in the UW to save the raffinose solution (Raffinose) (Adenosine), adenosine, citrate, succinate (Citrate) (Succinate), acetic acid (Ethanol), creatine / creatinine (creatine/creatinine), inosine (Inosine), lactic acid (Lactate) metabolites such as alanine. The content changes in different periods, and hypoxanthine (Allopurinol), glutathione (Glutathione), choline (Choline), and other metabolites had no obvious change of.2, 0h, 4h, 8h, 12h mixed group was analyzed by PCA and OPLS-DA. The UW Oh perfusion fluid samples have obvious aggregation, and 4h, 8h and 12h were separated from 0h, and more dispersed, from the PCA load diagram can be seen obvious difference is that, with the increase of the storage time of each group have obvious metabolic changes, but 4h, 8h, 12h between the sample hydrogen spectrum there is a certain overlap.3, in 4H with 8h, PCA and OPLS-DA analysis of pattern recognition between 4H and 12h, 8h and 12h, the metabolic differences between the groups, especially in the analysis of OPLS-DA pattern recognition show. Find out each group corresponding integral section difference of metabolic substances, but also affects the metabolism of transplanted liver Changes in biomarkers including succinate, citric acid, lactic acid, glutamine, inosine and adenine, glucose metabolism is caused by 4h, 8h, metabolite 12h and the reference point to the biggest difference. Conclusion 1, study on UW metabolism group 1HNMR method based on the change of endogenous small molecules to pass study on the metabolites of donor liver and UW preservation group transplantation normal group, to find the experiment that can represent the quality of donor liver transplantation using biomarkers to assess: succinate, citric acid, lactic acid, glutamine and inosine, to evaluate the quality of donor liver transplantation.2 assessment provides new ideas and methods these markers, anaerobic metabolism and glucose metabolism, three closely related to tricarboxylic acid cycle and adenine metabolism in human liver function proved important in The new supersedes the old..

【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R657.3

【參考文獻】

相關(guān)博士學位論文 前1條

1 林景超;化學性肝損傷及肝癌的代謝組學研究[D];上海交通大學;2008年

，

本文編號：1618725