【作者】 张丹丹；

【导师】 陈大忠；

【作者基本信息】 黑龙江中医药大学 ， 药剂学， 2017， 硕士

【摘要】 目的:运用超高效液相色谱联合四级杆飞行时间串联质谱(UPLC-Q-TOF-MS)的技术建立大鼠牛血清白蛋白(BSA)过敏反应模型的代谢组学方法,研究BSA和SHLI诱导过敏反应的大鼠尿液与正常生理状态下的尿液中内源性的小分子生物标志物的变化状况,探索与SHLI过敏反应有关的潜在小分子生物标志物,从而探讨SHLI过敏反应的代谢通路和过敏反应机制。方法:以BSA大鼠发生过敏反应为模型组,模拟大鼠发生过敏反应的过敏症状,实验将大鼠随机分为两组,即模型组和空白组,对大鼠的宏观体征表现进行考察,血清中类胰蛋白酶和组胺的含量、病理切片来综合评价动物模型的合理性;收集大鼠的尿液后,采用UPLC-Q-TOF-MS技术,对大鼠的尿液进行代谢组学分析,采集在正、负离子模式下的UPLC-MS图谱,经由Masslynx软件进行色谱峰的提取和匹配,根据主成分分析法(PCA)和正交偏最小二乘辨别分析(OPLS-DA)等方法分析BSA引发的过敏反应中内源性的小分子生物标志物的动态变化,从而筛选出与BS A过敏反应有关的潜在的内源性的小分子生物标志物。在前文建立代谢组学研究的基础上,对大鼠随机分为四组,即:空白组、SHLI高剂量组、SHLI中剂量组、SHLI低剂量组检测大鼠的组胺和类胰蛋白酶的含量、观察病理切片,运用代谢组学的方法对尿液进行测定分析,采用PC A和OPLS-DA的模式识别方法分析大鼠的代谢状况,解析与SHLI过敏反应相关的内源性的生物标志物和其代谢通路,在代谢水平上探索SHLI过敏反应的过敏机制。结果:本研究建立了基于UPLC-Q-TOF-MS技术研究大鼠过敏反应模型的代谢组学方法,大鼠在给予牛血清白蛋白后增加了血清中组胺和类胰蛋白酶的含量,同时结合大鼠的宏观体征表现和病理切片证明模型的成功性;本文进一步通过代谢组学的方法,观察到空白组和模型组具有较为明显的聚类分组,说明给予牛血清白蛋白后的大鼠的内源性生物标志物的含量发生了较大的改变,在代谢组学的水平上也证明过敏反应的动物模型造模成功,同时在大鼠过敏反应模型中鉴定了 14种与过敏反应相关的潜在的生物标志物,这些潜在的生物标志物可能主要与叶酸、色氨酸、异黄酮、烟酰胺和烟酸的代谢等通路有关。在SHLI的过敏反应中,与空白组比较,SHLI高、中剂量组的血清中组胺和类胰蛋白酶的含量显著增加,且具有显著性差异,SHLI低剂量组含量增加,但不具有显著性差异;病理切片显示:大鼠的肺和气管的病变程度均随着SHLI给药剂量的增加逐渐加重;基于建立的代谢组学的模型,发现SHLI处理组的代谢组学图谱发生了显著改变,SHLI组与空白组相比明显偏离,空白组和SHLI组在距离上存在剂量依赖性增加,且随着剂量的增加代谢分布波动越明显,鉴定了与SHLI诱导的过敏反应相关的15种潜在的生物标志物,这些生物标志物可能与亚麻酸、苯丙氨酸、嘌呤等的代谢通路有关。结论:本次研究第一次采用代谢组学的方法研究SHLI的过敏反应,建立了大鼠尿液代谢组学分析平台,探索SHLI诱导过敏反应的代谢通路,为SHLI的安全性研究奠定了基础,同时为TCMI的安全性研究与发展奠定一定的实验基础。更多还原

【Abstract】 Objective:We have established a metabolic method of rat for bovine serum albumin(BSA)induced by allergic reaction based on the technique of UPLC-Q-TOF-MS,to study the changes in endogenous metabolites between urines of rats in normal physiological conditions and bovine serum albumin,SHLI induced allergic reactions,identify potential biomarkers associated with allergic reactions,and then analyze the metabolic pathways and the metabolic mechanisms of allergic reactions induced by SHLI.METHODS:The bovine serum albumin-induced allergic reactions in rats were adopted as a model group,to simulate the symptoms of rat allergic reaction,The rats were randomly divided into two groups:control group and model group,we observed the systemic signs of performance in rats,the levels of tryptase and histamine in serum,,Pathological sections to comprehensively evaluate the rationality of animal models,then collected the rat urine,we used the UPLC-Q-TO F-MS to analyze the urine of rats,collected the UPLC-MS spectra in positive and negative ion mode,the chromatographic peaks were extracted and matched by Masslynx software,principal component analysis(PCA)and Partial Least Squares-Discriminant Analysis(PLS-DA)were used to analyze the dynamic changes of endogenous small molecule biomarkers in BSA-induced allergic reactions,to screen out the potential endogenous small molecule biomarkers associated with allergic reactions induced by BSA in the further.Based on the previously metabolomics model,The rats were randomly divided into four groups:control group,SHLI high dose group,SHLI medium dose group,SHLI low dose group,we tested the content of histamine and trypsin in rats,observed the pathological sections,then used metabolomics to analyze the urine of rats.The metabolic status of rats were analyzed by PCA and PLS-DA,analyzed the endogenous biomarkers and their metabolic pathways related to allergic reactions induced by SHLI,explore the allergic mechanism of SHLI allergic reactions at the metabolic level in the further.Results:This study established a metabolic method of rat allergic reaction model based on the UPLC-Q-TOF-MS technique,because of the administration of bovine serum albumin,the histamine and tryptase content has increased,at the same time,combined with the signs of the performance of rats and pathological sections has proved the success of the model;In this paper,by means of the metabolomics method in the further,we observed the control group and the model group have a more obvious clustering grouping,the content of endogenous bio markers in rats has a great changes after administration of bovine serum albumin,at the level of metabolomics also demonstrated that animal mode ls of allergic reactions were successful.14 kinds of potential biomarkers related to allergy were identified in the allergic reaction of rats model,these potential biomarkers may involve biosynthesis of isoflavone and folic acid and metabolism of tryptophan,nicotinic acid and nicotin-amide.In the allergic reaction of SHLI,compared to the control group,in the high dose group of SHLI and medium dose group of SHLI,the levels of histamine and tryptase were significantly increased and there was a significant difference,low dose group of SHLI has also increased,but there was not a significant difference.The pathological sections show that,degree of disease of the lung and trachea in rats has increased with the increase of the dosage.Based on the established metabolomics model,we found that the metabolomics map of SHLI groups has changed significantly,compared with the control group,the SHLI groups were significantly deviated,and there as a dose-dependent increase in distance between the control group and the SHLI high-dose group,and with the increase of the dose,the distribution of metabolic fluctuations were more obvious,identified 15 potential bio markers associated with SHLI-induced allergic reactions,these biomarkers may be associated with linolenic acid,phenylalanine,purine and other metabolic pathways.Conclusion:This study for the first time using metabolic methods to study the allergic reaction of SHLI,we established a urine metabolomics analysis platform of rats,to explore the mechanism of SHLI induced by allergic reactions,it laid the foundation for the safety research of SHLI and laid the experimental basis and method basis for the TCMI research at the same time.更多还原