【作者】 周鹏程；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2012， 硕士

【摘要】 第一部分猪急性肝衰竭过程中血清代谢物的动态变化模式目的：探索急性肝衰竭发生发展直至死亡过程中血清代谢物的动态变化模式,建立急性肝衰竭病情严重程度评估模型。方法：用D-半乳糖胺对猪(n=8)行急性肝衰竭造模,分别在给药前(0h)给药后18h、24h、36h,死亡前12h内取血清样本行超高效液相色谱-质谱检测。用Simca-P等软件进行多元统计分析,用Matlab进行模式识别和构建线性判别分析模型。结果：急性肝衰竭过程中血清代谢物至少存在3种上升的动态变化模式和1中下降的动态变化模式。每种模式在不同的时间段的变化趋势和程度不同。芳香族氨基酸(模式1)和溶血卵磷脂(模式4)可以作为急性肝衰竭严重程度的评估指标,而一些结合胆汁酸和长链肉毒碱(模式2)及甘氨胆酸(模式3)可以在早期检测到肝损伤。基于急性肝衰竭病情的严重程度可以在代谢组学水平上体现的事实,我们建立了包含苯丙氨酸和溶血卵磷脂18：1的线性判别分析模型来分期评估急性肝衰竭的严重程度。弃一法交叉验证显示该模型对于训练样本的判断准确率为91.67%,对于外部验证数据集的预测精确度为92.31%。结论：D-半乳糖胺诱导的猪急性肝衰竭发生发展直至死亡过程中血清代谢物至少有4种动态变化的模式,每种模式都有着相对独特的临床意义。急性肝衰竭病情的严重程度可以用代谢组学模型来评估。第二部分微囊悬浮型流化床式生物人工肝对急性肝衰竭猪血清代谢物的影响目的：从代谢组学角度揭示微囊悬浮型流化床式生物人工肝(FBBAL)治疗急性肝衰竭的机制,评估其安全性和疗效,以获得改进FBBAL的有用信息。方法：用D-半乳糖胺行急性肝衰竭造模,18h后21头猪分为以下3组分别处理6小时：FBBAL组,急性肝衰竭猪给予包含微囊化原代猪肝细胞的FBBAL治疗；假治疗组,急性肝衰竭猪给予包含空微囊的FBBAL治疗；肝衰竭对照组(n=7),不给予任何外部处理,只观察动物的一般状态。分别在给药前(0h)、给药后治疗前(18h)、治疗结束时(24h)和治疗结束后24h(48h)取血清样本行超高效液相色谱-质谱检测。用Simca-P和Matlab等软件进行多元统计分析。结果：FBBAL组的生存时间(70.4±11.5h)显著长于假治疗组(51.6±7.9h)肝衰竭对照组(49.3±6.6h)。治疗前(0h、18h)各组代谢谱没有明显不同。治疗后,FBBAL组的代谢谱和其他两组明显不同,并且后两组的代谢谱没有明显不同。经过FBBAL治疗胆汁酸进一步升高,而卵磷脂、溶血卵磷脂、脂肪酸和鞘磷脂进一步降低。结论：壳聚糖-海藻酸钠微囊对急性肝衰竭猪的血清代谢谱的短期影响较小。FBBAL具备一定的肝脏功能,能改变急性肝衰竭猪的血清代谢谱,从而延长受试猪的生存时间。然而FBBAL的作用不一定使得代谢物都靠近正常水平,其结构和功能仍需进一步完善。更多还原

【Abstract】 Part I Dynamic Patterns of Serum Metabolites in Aute Liver Failure PigsBackground&Aims:The study was aimed to explore the Dynamic Patterns of endogenous serum metabolites in the whole process of acute liver failure (ALF), and to develop a metabolomic model for evaluating the severity of ALF.Methods:ALF was induced in pigs (n=8) by intravenous administration of D-galactosamine. Serum samples were collected before the administration,18h,24h and36h after the administration and within12h before death and assayed by ultra performance liquid chromatography-mass spectrometry. The multivariate data was analyzed with SIMCA-P+12.0. Matlab Version7.8(R2009a) was used to perform pattern recognition analysis and construct a linear discriminant analysis model.Results:Three increased patterns and one decreased pattern were found in the whole process of ALF, and each pattern has its own distinct changing trends at different stages. Aromatic amino acids (Patternl) and Lysophosphatidylcholines (LPCs)(Pattern4) might be good markers for evaluating the severity of ALF, while some conjugated bile acids, long chain acylcarnitines (Pattern2) and Glycocholic acid (Pattern3) could indicate liver injury in the early stage. Inspired from the principal component analysis score plot that the pathogenetic condition of ALF aggravated with sampling time, a linear discriminant analysis (LDA) model based on phenylalanine and LPC18:1were further constructed for evaluating the severity of ALF. The leave-one-out cross-validation accuracy of91.67%for the training set and the prediction accuracy of92.31%for the external validation set confirmed its excellent performance.Conclusion:There are at least four Dynamic Patterns in the whole process of ALF, and each pattern has its own distinctive clinical significance. The severity of ALF could be evaluated by the metabolomics model.Part Ⅱ Effects of Fuidized Bed Bioartificial Liver on Serum Metabolites of Acute Liver Failure PigsBackground&Aims:Through characterizing the potential serum metablomics alterations of ALF pigs after the real and sham Fuidized Bed Bioartificial Liver (FBBAL) treatment, this study was aimed to reveal their therapy mechanism and evaluate their safety and get useful information for improving FBBAL.Methods:ALF was induced by intravenous administration of D-galactosamine.18hours later, pigs were treated for6hours as follows. In FBBAL group (B), pigs (n=7) were treated with FBBAL containing encapsulated primary porcine hepatocytes; in sham FBBAL group (S), pigs (n=7) were treated with FBBAL containing cell-free capsules; in ALF group (C), pigs (n=7) were only given intensive care. Serum samples were collected at0h,18h,24h and48h and assayed by ultra performance liquid chromatography-mass spectrometry. The multivariate data was analyzed with SIMCA-P+12.0.Results:Survival time was prolonged significantly (P<0.01) in the B group (70.4±11.5h) as compared with S group (51.6±7.9h) and C group (49.3±6.6h). No apparent metabolomics difference was observed between each group before FBBAL treatment, however, after the treatment, distinct clustering of B group and other two groups, which were intermixed, were presented. Phosphatidylcholines, lysophosphatidylcholines, sphingomyelinase and fatty acids were decreased further, while conjugated bile acids were increased further after FBBAL treatment.Conclusion:Alginate-chitosan capsules have no obvious influence on serum metabolites of ALF pigs. FBBAL possesses some liver functions and could alters the serum metabolome of ALF pigs, and ALF pigs survive longer thereby. Nevertheless, the alterations of metabolites are not universally toward health, and much should be done to improve the therapeutic effect of FBBAL.更多还原