脂质组学分析技术及其在发现免疫性疾病和病毒性肝炎相关生物标志物方面的研究

发布时间：2018-04-25 11:52

  本文选题：鞘脂组学 + 脂质组学　； 参考：《北京协和医学院》2014年博士论文

【摘要】：近年来,随着科学技术的发展与人们认识水平的提高,大量研究表明,以鞘脂为代表的功能性脂质类化合物不仅参与生物体内的能量代谢、构建细胞质膜结构,还具有广泛的生物学活性,譬如调控细胞增殖、分化、胞内交流和迁徙、胞间(或胞外)的信号传导、膜结构的转运、自噬和细胞凋亡等。因此,脂质类化合物的生物活性成为了近年来的研究热点。随着质谱技术的发展,特别是液相色谱联用质谱技术,脂质类化合物的结构信息被更多的揭示出来,其结构与功能的关系得到了更好的诠释,脂质组学也应运而生。脂质组学分析技术研究成为了分析化学领域的热点。 内源性的脂质类化合物在机体内的天然丰度差别非常大,生源丰度通常从纳克级到微克级不等。其中,鞘脂类化合物通常在纳克级水平,且离子化效率较低,属于比较难检测的一类化合物。鉴于脂质类化合物自然丰度差异巨大,结合其结构特点,我们采用了不同类型的质谱来研究脂质分析技术,构建了一套组合式的脂质分析平台。本论文首先基于鞘脂代谢网络,采用高效液相色谱串联三重四级杆质谱(HPLC-MS/MS),建立了一套靶向鞘脂质组学的定量分析方法,能够同时分析43个鞘脂代谢网络核心化合物。本论文首先优化了生物样本(血浆、血清或组织匀浆液,0.1mL)前处理方法,采用甲基叔丁基醚-甲醇-水(20：6：5,v/v/v)三元混合溶剂系统对生物样本(血浆、血清或组织匀浆液,0.1mL)进行脂质提取,提取回收率达到60%-80%。液相色谱分析采用反相C8色谱柱,流动相分别为,A相：0.2%甲酸2.0mM甲酸铵水溶液,B相：0.2%甲酸1.0mM甲酸铵甲醇溶液,梯度洗脱模式,质谱检测采用ESI正离子分段多反应离子监测模式,分段检测的引入显著提高了平台的灵敏度。每一个检测分段内均至少有一个非内源性同系物作为内标来保证定量准确性。方法验证表明,最低定量限：1.0pmol/mg protein(所有鞘脂)；线性范围：12.5-2000.0pmol/mg protein(鞘磷脂类)、2.5-400.0pmol/mg protein(其他鞘脂类)；线性相关系数：r0.99；日内日间精密度均小于15%；准确度：80%-120%;工作溶液室温放置6小时和-20℃放置60天稳定。上述结果证明方法准确可靠,适合常规生物样品分析。为了更全面的使方法覆盖鞘脂代谢网络的核心化合物,我们依据鞘脂类化合物的结构和质谱裂解规律,推演并增加了多反应监测的离子对的数目,扩充了鞘脂组学分析方法所监测的目标化合物的数量从43个到74个。同时,我们引入了高效液相色谱联用高分辨质谱仪——傅里叶变换离子回旋共振质谱仪(HPLC-LTQ-FTICRMS)建立一套适用于分析高丰度脂质的脂质组学定量分析平台,液相色谱分析采用反相C8色谱柱,流动相分别为,A相：2mM乙酸铵缓冲盐水溶液含有0.1%甲酸,B相：含有2mM乙酸铵和0.1%甲酸的异丙醇／乙腈(2：5,v/v)溶液,梯度洗脱模式。ESI正离子高分辨全扫描模式监测,利用一级高分辨质谱数据,我们采用Lipid Data Analyzer(?)软件对数据进行高内涵高通量的处理。该平台可以同时监测4大类,包括甘油三酯、甘油二酯、甘油磷脂酰胆碱、甘油磷脂酰乙醇胺合计216种脂质类化合物。部分方法验证表明：最低定量限：0.02nmol/mg protein(所有脂质)；线性范围：0.02-200nmol/mL;线性相关系数：r0.95；精密度小于15%,准确度：80%-120%。上述结果证明该平台适合用于生物样品靶向定量脂质组学研究。具有使用样品量少、高内涵、高通量,并具有同时定性和定量分析的特点。 在进行脂质分析技术平台建立过程中,我们将所建立的分析方法及时应用于实际生物样本的分析以及筛选与疾病相关生物标志物的研究中。首先,我们将鞘脂分析技术用于了寻找2,4-二硝基氟苯诱发的迟发型超敏反应模型小鼠的血浆、肾脏、肝脏和脾脏中与模型相关的生物标志物,及模型小鼠给予治疗剂量雷公藤甲素后的与药效相关的生物标志物。经过所建立鞘脂组学方法分析后,我们对得到的鞘脂组学数据进行了统计分析,发现了23个鞘脂类化合物(12个神经酰胺类化合物,3个鞘胺醇类化合物,3个糖基化神经酰胺类化合物和5个神经鞘磷脂类化合物)为迟发超敏反应和雷公藤甲素治疗的潜在生物标志物。同时,我们发现雷公藤甲素使得迟发超敏反应模型小鼠的肾脏中10个鞘脂类化合物的代谢发生显著变化,可能与雷公藤甲素潜在的肾毒性相关。该研究表明,靶向定量鞘脂组学结合多变量统计方法能够在生物样品中有效的筛选潜在的鞘脂生物标志物。 已有文献研究结果表明肝炎病毒和丙型肝炎病毒侵入、复制、转录和出芽过程均与鞘脂密切相关,因此本论文首次开展了乙肝及其相关慢性肝病的鞘脂组学研究,利用鞘脂组学分析技术,分析慢性乙肝患者血清中的鞘脂类化合物。该研究总共使用了两个临床队列,合计156例血清样本,由北京佑安医院提供。每个临床队列分为健康对照组、慢乙肝组和慢乙肝导致的慢加急性肝衰竭组(HBV-ACLF)。结果表明,鞘脂代谢网络的紊乱与疾病的发展密切相关,发现并确认了9个鞘脂生物标志物,为慢性乙肝疾病发病机理研究提供线索。此外,发现了血清中dhCer(d18:0/24:0)水平的降低预示着HBV-ACLF患者预后不良,提供了一种新的方法评估HBV-ACLF的预后,进而有助于优化治疗方案,选择保守治疗或者尽早肝移植。 随着脂质组学分析技术平台的建立完善,我们将脂质组学分析技术应用于研究慢性丙肝患者的血浆脂质组。患者血浆样品由北京佑安医院提供。包括113例慢性丙肝患者和11例健康受试者,其中患者组根据其肝穿活检结果显示的肝内炎症级别分为IG01,IG2,IG34三组。结果表明,各疾病组与健康受试者之间的血浆脂质水平差异显著(检出的117个脂质中,47个脂质水平显著改变),而患者中不同炎症组之间差异较小(只有8个脂质水平显著改变)。说明血浆脂质组的改变与疾病的发生更相关,而不是炎症发展程度。丙肝慢性感染导致的营养不良很可能是一个重要原因。HexCer(d18:1/22:0)、HexCer (d18:1/24:1)、HexCer (d18:1/24:0)、PC(34:4)和PC(40：5)在轻度和重度肝内炎症组之间存在显著差异,显示它们可作为评价肝内炎症的潜在非侵入性指标。 本论文研究后期探索性的建立脂质组学整体定性筛查和靶向定量分析新技术,首次建立了一套在线串联二维(HILIC×RP)超高效液相色谱串联三重四级杆质谱仪脂质组学平台。三步筛选脂质化合物的策略充分展示了平台的对于各种生物样品都具有普适性,后续的使用MRM模式对筛选出来的化合物进行定量展示了平台准确定量的能力。尤其是该平台可以在线同时区分并准确定量PC和SM类化合物,使得需要前处理去除两类化合物测定干扰的问题得以解决,提高了测定的效率和准确性。我们进一步将所建立的平台用于大鼠血浆脂质组的分析,其中包括雄性和雌性大鼠的血浆和采集于不同位置的血浆(眼缘静脉、腹主动脉、尾静脉)。结果表明,所建立的方法可以同时定量154个脂质类化合物,包括1个ceramide,1个ceramide-1P,1个sphingosine,60个PC,19个SM和72个TG。统计分析表明,雄性和雌性大鼠血浆脂质组差异显著。来自不同取血位置的血浆中的个别脂质也存在显著差异。上述结果可以给我们一个启示,即当我们去重复或者验证一个脂质组学的研究时,重复同样的样品采集方法非常重要,不仅包括实验动物的性别还有样品的采集部位。 本论文建立了覆盖脂质代谢网络核心脂质的整体定性和靶向定量分析平台,通过分析实际生物样品,充分展示了方法的深度和广度,并将分析结果与生物学意义相关联,表明生命体的脂质组与疾病发生发展息息相关,为疾病的诊断、预后以及疾病机理的研究奠定了基础。由于本论文的研究均属于单一时间点的截面研究类型,无法动态关注脂质的代谢转化,今后可采用药代动力学的原理和手段关注这一方面。
[Abstract]:In recent years, with the development of science and technology and the improvement of people's understanding level, a large number of studies have shown that functional liposomes, represented by sphingolipids, not only participate in the energy metabolism in the organism, construct the cell membrane structure, but also have extensive biological activity, such as cell proliferation, differentiation, intracellular communication and migration, and intercellular (or intercellular). The signal transduction, transport of membrane structure, autophagy and apoptosis, etc. Therefore, the biological activity of liposomes has become a hot topic in recent years. With the development of mass spectrometry technology, especially liquid chromatography coupled mass spectrometry, the structure information of liposomes has been more revealed, and its structure and function are related to With better interpretation, lipid omics has also emerged. Lipid group analysis technology has become a hot topic in analytical chemistry.
The natural abundances of endogenous lipids are very large in the body, and the abundance of the sources usually varies from NAC to microgram. Among them, the sphingolipids are usually at the NAC level and are less ionized, and are relatively difficult to detect. We use different types of mass spectrometry to study lipid analysis techniques and construct a set of combined lipid analysis platform. Firstly, based on the sphingolipid metabolic network, we set up a set of quantitative analysis methods for target sheath liposomes by using high performance liquid chromatography (HPLC) and three weight four level mass spectrometry (HPLC-MS/MS). This paper first optimized the pretreatment methods of biological samples (plasma, serum or tissue homogenate, 0.1mL), using methyl tert butyl ether methanol water (20:6:5, v/v/v) three element mixed solvent system to extract the lipid from the biological samples (plasma, serum or tissue homogenate, 0.1mL) and extract the recovery rate in this paper. The reversed phase C8 chromatographic column was used for 60%-80%. liquid chromatography analysis. The mobile phase was respectively, A phase, 0.2% formic acid 2.0mM formate aqueous solution, B phase: 0.2% formic acid 1.0mM formate methanol solution, gradient elution mode, and mass spectrometric detection using ESI positive ion subsection multi reaction ion monitoring mode. The introduction of segmented detection improved the sensitivity of the platform significantly. Each detection section has at least one non endogenous homologue as an internal standard to ensure quantitative accuracy. Method validation shows that the minimum quantitative limit is 1.0pmol/mg protein (all sphingolipids); linear range: 12.5-2000.0pmol/mg protein (sphingomyelin), 2.5-400.0pmol/ mg protein (other sphingolipids); linear correlation coefficient: r0.99 The daily intraday precision is less than 15%, the accuracy is 80%-120%, the working solution is placed at room temperature for 6 hours and at -20 C for 60 days. The results show that the method is accurate and reliable, suitable for the analysis of conventional biological samples. The law of mass spectrometry has deduced and increased the number of ion pairs in the multi reaction monitoring, and expanded the number of target compounds monitored by the sphingolipid analysis method from 43 to 74. At the same time, we introduced a high resolution high resolution mass spectrometer with high performance liquid chromatography (high resolution mass spectrometry), Fu Liye transformation and cyclotron resonance mass spectrometer (HPLC-LTQ-FTICRMS). It is suitable for the analysis of liposome quantitative analysis platform for high abundance lipids. The liquid chromatography analysis uses reverse phase C8 column, A phase, respectively, 2mM ammonium acetate buffer salt solution contains 0.1% formic acid, B phase: 2mM ammonium acetate and 0.1% formic acid isopropanol / acetonitrile (2:5, v/v) solution, gradient elution mode.ESI positive ion resolution Full scan mode monitoring, using the first class high resolution mass spectrometry data, we use Lipid Data Analyzer (?) software to carry out high and high throughput processing. The platform can simultaneously monitor 4 major categories, including triglycerides, triglycerides, glycerin phosphatidylcholine, glycerin phospholipid ethanolamine, 216 kinds of liposomes. The minimum quantitative limit: 0.02nmol/mg protein (all lipids); linear range: 0.02-200nmol/mL; linear correlation coefficient: r0.95; precision less than 15%, accuracy: the 80%-120%. results show that the platform is suitable for the target quantitative liposome study of biological samples. It has the characteristics of simultaneous qualitative and quantitative analysis.
In the course of the establishment of the lipid analysis platform, we applied the analytical method in time to the analysis of actual biological samples and in the screening of biomarkers related to disease. First, we used the sphingolipid analysis technique to find the plasma of the delayed hypersensitivity model mice induced by 2,4- two nitrofluorrobenzene. The biomarkers associated with the model in the kidney, liver and spleen, and model mice were given the biological markers associated with the therapeutic dose of Tripterygium wilfordii. After the analysis of the sphingolipids, we analyzed the obtained sphingolipid data and found 23 sphingolipids (12 ceramide compounds). Class compounds, 3 sheathing amine alcohols, 3 glycosylated ceramides and 5 nerve sphingomyelin compounds are potential biomarkers of delayed hypersensitivity and Tripterygium wilfordii treatment. Meanwhile, we found that triptolide makes 10 sphingolipids in the kidneys of delayed hypersensitive mice. Significant changes in life may be associated with potential nephrotoxicity of triptolide. This study suggests that targeted quantitative sphingolipids combined with multivariate statistical methods can effectively screen potential sphingolipid biomarkers in biological samples.
Studies have shown that the invasion, replication, transcription and buds of hepatitis C virus and hepatitis C virus are closely related with sphingolipids. Therefore, the study of sphingolipids in hepatitis B and its related chronic liver diseases was first carried out in this paper, and the sphingolipid compounds in sera of patients with chronic hepatitis B were analyzed by means of sphingolipid analysis. A total of two clinical queues were used, a total of 156 serum samples were provided by the Beijing you an hospital. Each clinical cohort was divided into a healthy control group, a slow hepatitis B group and a slow and chronic hepatitis B induced chronic liver failure group (HBV-ACLF). The results showed that the disorders of the sphingolipid metabolic network were closely related to the development of the disease, and 9 sphingolipids were identified and confirmed. Biomarkers provide clues for the pathogenesis of chronic hepatitis B disease. In addition, the decrease of dhCer (d18:0/24:0) levels in serum indicates that the prognosis of HBV-ACLF patients is poor. A new method is provided to evaluate the prognosis of HBV-ACLF, and thus help to optimize the treatment case, select conservative treatment or early liver transplantation.
With the establishment and perfection of the liposome analysis technique platform, we applied liposome analysis technique to the study of plasma lipid groups in chronic hepatitis C patients. The plasma samples were provided by the Beijing you an hospital, including 113 patients with chronic hepatitis C and 11 healthy subjects, among which the patient group was based on liver biopsy results. The level of the disease was divided into groups of IG01, IG2, and IG34 three. The results showed that the plasma lipid levels of each disease group were significantly different from those in the healthy subjects (117 lipids, 47 lipid levels were significantly changed), and the difference between the different inflammatory groups was small (only 8 lipid levels were significantly changed). .HexCer (d18:1/22:0), HexCer (d18:1/24:1), HexCer (d18:1/24:0), PC (34:4) and PC (40:5) have significant differences between mild and severe intrahepatic inflammation groups, indicating that they can be used as a potential evaluation of intrahepatic inflammation. In non invasive indicators.
In this paper, we set up a new technology for lipid omics and target quantitative analysis, and first established a series of online series two-dimensional (HILIC * RP) super high performance liquid chromatography series three weight four level mass spectrometer liposome platform. The strategy of screening lipid compounds in three steps fully demonstrated the platform for various organisms. The samples are universally suitable, and the subsequent use of MRM model to quantitatively demonstrate the accuracy and quantitative ability of the selected compounds. In particular, the platform can distinguish and accurately quantify PC and SM compounds on line, making the problem of removing two kinds of compounds determined by preprocessing and improving the effectiveness of the determination. Rate and accuracy. We further used the established platform for the analysis of rat plasma lipid groups, including the plasma of male and female rats and plasma collected at different positions (ophthalmic vein, abdominal aorta, tail vein). The results showed that the established method could quantify 154 lipids, including 1 ceramide and 1. Ceramide-1P, 1 sphingosine, 60 PC, 19 SM and 72 TG. statistical analysis showed that the plasma lipid groups of male and female rats were significantly different. There were significant differences in the individual lipids from different blood fetching positions. The results can give us a revelation, when we repeat or verify a liposome study, It is very important to repeat the same sample collection method, including not only the sex of experimental animals, but also the location of sample collection.
In this paper, the overall qualitative and target quantitative analysis platform covering lipid metabolism network core lipid is established. By analyzing the actual biological samples, the depth and breadth of the method are fully demonstrated, and the analysis results are related to the biological significance. It shows that the lipid groups in the life body are closely related to the development of the disease, and the diagnosis of the disease is predefined. After the study of the disease mechanism, the research of this paper belongs to the type of single time point, which can not dynamically pay attention to the metabolic transformation of lipid. In the future, we can use the principle and means of pharmacokinetics to pay attention to this aspect.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R593.2;R512.6
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本文编号：1801191