基于~1HNMR的代谢组学技术在移植供肝质量和功能评估中的临床实验研究

发布时间：2018-03-16 06:31

本文选题：移植供肝　切入点：代谢组学　出处：《新乡医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的本研究拟以核磁共振氢谱(1H nuclear magnetic resonance,1HNMR)代谢组学技术为基础,在临床实际中建立移植肝脏保存模型,对移植肝脏低温保存中不同时间节点的代谢组进行研究,分析在低温保存中的代谢变化,建立以核磁共振氢谱(1Hnuclear magnetic resonance,1HNMR)代谢组学在移植供肝质量和功能评估中新方法。方法(1)随机选取40例低温保存移植肝脏,供体年龄控制在50岁以内,性别随机分配。根据离体肝脏保存的不同时间(基点Oh、4h、8h、12h)每例取保存液2ml,每个时间点10例样本,收集到的UW液应自门静脉原位灌注后,至肝脏颜色变为土黄色,从上下腔静脉和下下腔静脉流出收集,收集后立即放置液氮中保存以备使用。首先对采集到样本制备,然后使用Varian UNITY INOVA 600 MHz超导脉冲傅立叶变换核磁共振谱仪采集保存液样本和组织提取物的核磁共振数据(NMR)。NMR图谱是由自由感应衰减(free induction decay,FID)信号经超导脉冲傅立叶转换而来,最后所有的数据输出并保存,用于模式识别分析。把保存的数据先由Pareto标度化(Pareto scaling)和平均中心化(mean centering)进行预处理,再由 SMCA-P12.0软件(v12.0, Umetrics, Ume,Sweden)将得到的数据采用主成分分析法(principal component analysis, PCA)分析,为强化组间差异,进一步采用偏最小二乘法判别分析(PLS-DA)或正交信号校正(OSC)分析。以得分图(scores plot)和因子载荷图(loadings plot)的形式表示分析结果。四组保存液样本代谢物的归一化积分值采用统计学SPSS 17.0对单因素方差分析(ANOVA),P0.05认为差异具有统计学意义。(2)建立以1HNMR代谢组学技术为基础的肝脏保存液代谢组学平台,探讨1HNMR技术在移植肝脏保存中功能评估和质量评估应用的可行性,并通过UW液的在不同时期的代谢变化,同时找出引起这些差异变化的主要代谢物,作为潜在的标志物。结果1、1H-NMR谱图中可以看出,UW保存液中棉子糖(Raffinose)、腺苷(Adenosine)、柠檬酸盐(Citrate)、琥珀酸盐(Succinate)、乙酸(Ethanol)、肌酸/肌酐(creatine/creatinine)、次黄苷(Inosine)、乳酸(Lactate)丙氨酸、等代谢物的含量在不同时期发生了变化,而次黄嘌呤(Allopurinol)、谷胱甘肽(Glutathione)、胆碱(Choline)、等代谢物则变化不明显。2、对0h、4h、8h、12h混合组先进行了PCA和OPLS-DA分析。其中Oh灌注的UW液样本有明显的聚集情况,而4h、8h及12h明显的与0h分开,且较为分散,从PCA载荷图是可以看出明显的差别的,说明随着保存时间的延长,各组都发生了明显的代谢变化,是但4h、8h、12h样本之间的氢谱图之间存在一定重叠。3、在4h与8h、4h与12h以及8h与12h之间进行了PCA和OPLS-DA模式识别分析,各组之间代谢差异明显,特别是显现在OPLS-DA模式识别分析。找出每组之间差异明显的积分区段所对应的代谢物质,也是影响着移植供肝代谢变化明显的生物标志物。其中琥珀酸盐、柠檬酸盐、乳酸、谷氨酰胺以及次黄苷,腺嘌呤、葡萄糖的代谢是引起4h、8h、12h组与基准点差异最大的代谢物。结论1、基于1HNMR的UW液代谢组学研究方法可以通过研究正常组和保存组移植供肝UW液的小分子内源性代谢物的变化,找到能够实验中发现能够代表移植供肝质量用以评估的生物标志物：琥珀酸盐、柠檬酸盐、乳酸、谷氨酰胺以及次黄苷等,为评价移植供肝质量评估提供新的思路和方法。2、这些标志物与糖代谢的无氧酵解,三羧酸循环及腺嘌呤代谢密切相关,证实肝脏在人体的新陈代谢中重要的作用。
[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..

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.3

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