本文選題：鞘脂組學(xué) + 脂質(zhì)組學(xué)��； 參考：《北京協(xié)和醫(yī)學(xué)院》2014年博士論文

【摘要】：近年來(lái),隨著科學(xué)技術(shù)的發(fā)展與人們認(rèn)識(shí)水平的提高,大量研究表明,以鞘脂為代表的功能性脂質(zhì)類化合物不僅參與生物體內(nèi)的能量代謝、構(gòu)建細(xì)胞質(zhì)膜結(jié)構(gòu),還具有廣泛的生物學(xué)活性,譬如調(diào)控細(xì)胞增殖、分化、胞內(nèi)交流和遷徙、胞間(或胞外)的信號(hào)傳導(dǎo)、膜結(jié)構(gòu)的轉(zhuǎn)運(yùn)、自噬和細(xì)胞凋亡等。因此,脂質(zhì)類化合物的生物活性成為了近年來(lái)的研究熱點(diǎn)。隨著質(zhì)譜技術(shù)的發(fā)展,特別是液相色譜聯(lián)用質(zhì)譜技術(shù),脂質(zhì)類化合物的結(jié)構(gòu)信息被更多的揭示出來(lái),其結(jié)構(gòu)與功能的關(guān)系得到了更好的詮釋,脂質(zhì)組學(xué)也應(yīng)運(yùn)而生。脂質(zhì)組學(xué)分析技術(shù)研究成為了分析化學(xué)領(lǐng)域的熱點(diǎn)。 內(nèi)源性的脂質(zhì)類化合物在機(jī)體內(nèi)的天然豐度差別非常大,生源豐度通常從納克級(jí)到微克級(jí)不等。其中,鞘脂類化合物通常在納克級(jí)水平,且離子化效率較低,屬于比較難檢測(cè)的一類化合物。鑒于脂質(zhì)類化合物自然豐度差異巨大,結(jié)合其結(jié)構(gòu)特點(diǎn),我們采用了不同類型的質(zhì)譜來(lái)研究脂質(zhì)分析技術(shù),構(gòu)建了一套組合式的脂質(zhì)分析平臺(tái)。本論文首先基于鞘脂代謝網(wǎng)絡(luò),采用高效液相色譜串聯(lián)三重四級(jí)桿質(zhì)譜(HPLC-MS/MS),建立了一套靶向鞘脂質(zhì)組學(xué)的定量分析方法,能夠同時(shí)分析43個(gè)鞘脂代謝網(wǎng)絡(luò)核心化合物。本論文首先優(yōu)化了生物樣本(血漿、血清或組織勻漿液,0.1mL)前處理方法,采用甲基叔丁基醚-甲醇-水(20：6：5,v/v/v)三元混合溶劑系統(tǒng)對(duì)生物樣本(血漿、血清或組織勻漿液,0.1mL)進(jìn)行脂質(zhì)提取,提取回收率達(dá)到60%-80%。液相色譜分析采用反相C8色譜柱,流動(dòng)相分別為,A相：0.2%甲酸2.0mM甲酸銨水溶液,B相：0.2%甲酸1.0mM甲酸銨甲醇溶液,梯度洗脫模式,質(zhì)譜檢測(cè)采用ESI正離子分段多反應(yīng)離子監(jiān)測(cè)模式,分段檢測(cè)的引入顯著提高了平臺(tái)的靈敏度。每一個(gè)檢測(cè)分段內(nèi)均至少有一個(gè)非內(nèi)源性同系物作為內(nèi)標(biāo)來(lái)保證定量準(zhǔn)確性。方法驗(yàn)證表明,最低定量限：1.0pmol/mg protein(所有鞘脂)；線性范圍：12.5-2000.0pmol/mg protein(鞘磷脂類)、2.5-400.0pmol/mg protein(其他鞘脂類)；線性相關(guān)系數(shù)：r0.99；日內(nèi)日間精密度均小于15%；準(zhǔn)確度：80%-120%;工作溶液室溫放置6小時(shí)和-20℃放置60天穩(wěn)定。上述結(jié)果證明方法準(zhǔn)確可靠,適合常規(guī)生物樣品分析。為了更全面的使方法覆蓋鞘脂代謝網(wǎng)絡(luò)的核心化合物,我們依據(jù)鞘脂類化合物的結(jié)構(gòu)和質(zhì)譜裂解規(guī)律,推演并增加了多反應(yīng)監(jiān)測(cè)的離子對(duì)的數(shù)目,擴(kuò)充了鞘脂組學(xué)分析方法所監(jiān)測(cè)的目標(biāo)化合物的數(shù)量從43個(gè)到74個(gè)。同時(shí),我們引入了高效液相色譜聯(lián)用高分辨質(zhì)譜儀——傅里葉變換離子回旋共振質(zhì)譜儀(HPLC-LTQ-FTICRMS)建立一套適用于分析高豐度脂質(zhì)的脂質(zhì)組學(xué)定量分析平臺(tái),液相色譜分析采用反相C8色譜柱,流動(dòng)相分別為,A相：2mM乙酸銨緩沖鹽水溶液含有0.1%甲酸,B相：含有2mM乙酸銨和0.1%甲酸的異丙醇／乙腈(2：5,v/v)溶液,梯度洗脫模式。ESI正離子高分辨全掃描模式監(jiān)測(cè),利用一級(jí)高分辨質(zhì)譜數(shù)據(jù),我們采用Lipid Data Analyzer(?)軟件對(duì)數(shù)據(jù)進(jìn)行高內(nèi)涵高通量的處理。該平臺(tái)可以同時(shí)監(jiān)測(cè)4大類,包括甘油三酯、甘油二酯、甘油磷脂酰膽堿、甘油磷脂酰乙醇胺合計(jì)216種脂質(zhì)類化合物。部分方法驗(yàn)證表明：最低定量限：0.02nmol/mg protein(所有脂質(zhì))；線性范圍：0.02-200nmol/mL;線性相關(guān)系數(shù)：r0.95；精密度小于15%,準(zhǔn)確度：80%-120%。上述結(jié)果證明該平臺(tái)適合用于生物樣品靶向定量脂質(zhì)組學(xué)研究。具有使用樣品量少、高內(nèi)涵、高通量,并具有同時(shí)定性和定量分析的特點(diǎn)。 在進(jìn)行脂質(zhì)分析技術(shù)平臺(tái)建立過(guò)程中,我們將所建立的分析方法及時(shí)應(yīng)用于實(shí)際生物樣本的分析以及篩選與疾病相關(guān)生物標(biāo)志物的研究中。首先,我們將鞘脂分析技術(shù)用于了尋找2,4-二硝基氟苯誘發(fā)的遲發(fā)型超敏反應(yīng)模型小鼠的血漿、腎臟、肝臟和脾臟中與模型相關(guān)的生物標(biāo)志物,及模型小鼠給予治療劑量雷公藤甲素后的與藥效相關(guān)的生物標(biāo)志物。經(jīng)過(guò)所建立鞘脂組學(xué)方法分析后,我們對(duì)得到的鞘脂組學(xué)數(shù)據(jù)進(jìn)行了統(tǒng)計(jì)分析,發(fā)現(xiàn)了23個(gè)鞘脂類化合物(12個(gè)神經(jīng)酰胺類化合物,3個(gè)鞘胺醇類化合物,3個(gè)糖基化神經(jīng)酰胺類化合物和5個(gè)神經(jīng)鞘磷脂類化合物)為遲發(fā)超敏反應(yīng)和雷公藤甲素治療的潛在生物標(biāo)志物。同時(shí),我們發(fā)現(xiàn)雷公藤甲素使得遲發(fā)超敏反應(yīng)模型小鼠的腎臟中10個(gè)鞘脂類化合物的代謝發(fā)生顯著變化,可能與雷公藤甲素潛在的腎毒性相關(guān)。該研究表明,靶向定量鞘脂組學(xué)結(jié)合多變量統(tǒng)計(jì)方法能夠在生物樣品中有效的篩選潛在的鞘脂生物標(biāo)志物。 已有文獻(xiàn)研究結(jié)果表明肝炎病毒和丙型肝炎病毒侵入、復(fù)制、轉(zhuǎn)錄和出芽過(guò)程均與鞘脂密切相關(guān),因此本論文首次開(kāi)展了乙肝及其相關(guān)慢性肝病的鞘脂組學(xué)研究,利用鞘脂組學(xué)分析技術(shù),分析慢性乙肝患者血清中的鞘脂類化合物。該研究總共使用了兩個(gè)臨床隊(duì)列,合計(jì)156例血清樣本,由北京佑安醫(yī)院提供。每個(gè)臨床隊(duì)列分為健康對(duì)照組、慢乙肝組和慢乙肝導(dǎo)致的慢加急性肝衰竭組(HBV-ACLF)。結(jié)果表明,鞘脂代謝網(wǎng)絡(luò)的紊亂與疾病的發(fā)展密切相關(guān),發(fā)現(xiàn)并確認(rèn)了9個(gè)鞘脂生物標(biāo)志物,為慢性乙肝疾病發(fā)病機(jī)理研究提供線索。此外,發(fā)現(xiàn)了血清中dhCer(d18:0/24:0)水平的降低預(yù)示著HBV-ACLF患者預(yù)后不良,提供了一種新的方法評(píng)估HBV-ACLF的預(yù)后,進(jìn)而有助于優(yōu)化治療方案,選擇保守治療或者盡早肝移植。 隨著脂質(zhì)組學(xué)分析技術(shù)平臺(tái)的建立完善,我們將脂質(zhì)組學(xué)分析技術(shù)應(yīng)用于研究慢性丙肝患者的血漿脂質(zhì)組。患者血漿樣品由北京佑安醫(yī)院提供。包括113例慢性丙肝患者和11例健康受試者,其中患者組根據(jù)其肝穿活檢結(jié)果顯示的肝內(nèi)炎癥級(jí)別分為IG01,IG2,IG34三組。結(jié)果表明,各疾病組與健康受試者之間的血漿脂質(zhì)水平差異顯著(檢出的117個(gè)脂質(zhì)中,47個(gè)脂質(zhì)水平顯著改變),而患者中不同炎癥組之間差異較小(只有8個(gè)脂質(zhì)水平顯著改變)。說(shuō)明血漿脂質(zhì)組的改變與疾病的發(fā)生更相關(guān),而不是炎癥發(fā)展程度。丙肝慢性感染導(dǎo)致的營(yíng)養(yǎng)不良很可能是一個(gè)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)在輕度和重度肝內(nèi)炎癥組之間存在顯著差異,顯示它們可作為評(píng)價(jià)肝內(nèi)炎癥的潛在非侵入性指標(biāo)。 本論文研究后期探索性的建立脂質(zhì)組學(xué)整體定性篩查和靶向定量分析新技術(shù),首次建立了一套在線串聯(lián)二維(HILIC×RP)超高效液相色譜串聯(lián)三重四級(jí)桿質(zhì)譜儀脂質(zhì)組學(xué)平臺(tái)。三步篩選脂質(zhì)化合物的策略充分展示了平臺(tái)的對(duì)于各種生物樣品都具有普適性,后續(xù)的使用MRM模式對(duì)篩選出來(lái)的化合物進(jìn)行定量展示了平臺(tái)準(zhǔn)確定量的能力。尤其是該平臺(tái)可以在線同時(shí)區(qū)分并準(zhǔn)確定量PC和SM類化合物,使得需要前處理去除兩類化合物測(cè)定干擾的問(wèn)題得以解決,提高了測(cè)定的效率和準(zhǔn)確性。我們進(jìn)一步將所建立的平臺(tái)用于大鼠血漿脂質(zhì)組的分析,其中包括雄性和雌性大鼠的血漿和采集于不同位置的血漿(眼緣靜脈、腹主動(dòng)脈、尾靜脈)。結(jié)果表明,所建立的方法可以同時(shí)定量154個(gè)脂質(zhì)類化合物,包括1個(gè)ceramide,1個(gè)ceramide-1P,1個(gè)sphingosine,60個(gè)PC,19個(gè)SM和72個(gè)TG。統(tǒng)計(jì)分析表明,雄性和雌性大鼠血漿脂質(zhì)組差異顯著。來(lái)自不同取血位置的血漿中的個(gè)別脂質(zhì)也存在顯著差異。上述結(jié)果可以給我們一個(gè)啟示,即當(dāng)我們?nèi)ブ貜?fù)或者驗(yàn)證一個(gè)脂質(zhì)組學(xué)的研究時(shí),重復(fù)同樣的樣品采集方法非常重要,不僅包括實(shí)驗(yàn)動(dòng)物的性別還有樣品的采集部位。 本論文建立了覆蓋脂質(zhì)代謝網(wǎng)絡(luò)核心脂質(zhì)的整體定性和靶向定量分析平臺(tái),通過(guò)分析實(shí)際生物樣品,充分展示了方法的深度和廣度,并將分析結(jié)果與生物學(xué)意義相關(guān)聯(lián),表明生命體的脂質(zhì)組與疾病發(fā)生發(fā)展息息相關(guān),為疾病的診斷、預(yù)后以及疾病機(jī)理的研究奠定了基礎(chǔ)。由于本論文的研究均屬于單一時(shí)間點(diǎn)的截面研究類型,無(wú)法動(dòng)態(tài)關(guān)注脂質(zhì)的代謝轉(zhuǎn)化,今后可采用藥代動(dòng)力學(xué)的原理和手段關(guān)注這一方面。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R593.2;R512.6
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本文編號(hào)：1801191