【作者】 李昕；

【导师】 贾伟；

【作者基本信息】 上海交通大学 ， 药理学， 2010， 博士

【摘要】 出生缺陷是一种在婴儿出生以前在母体子宫内便发生的身体结构上的发育异常。它包括由于胚胎发育紊乱引起的形态、结构、功能、代谢、精神行为等方面的异常情况。随着人口增长和疾病模式的转变,出生缺陷现象日益突出,已成为世界上婴儿死亡、儿童和成人致残的主要原因之一。我国现有的5100多万残疾人和2200多万的各种遗传病患者中,大部分致残原因为出生缺陷,这给国家和社会带来了极大的经济负担。导致出生缺陷的原因非常复杂,许多常见的出生缺陷是多因素疾病,既包括染色体和基因异常等遗传性缺陷,又包括环境因素(营养,药物,环境污染)导致造成的缺陷。寻找和出生缺陷相关的特异性生物标记物成为目前该疾病诊断和治疗研究的热点与难点。人们除了对在孕妇产前补充叶酸这一预防出生缺陷的手段外,还难以找到其它预防和治疗出生缺陷的有效手段。目前出生缺陷产前诊断的方法主要包括影像学(超声、CT)及临床生化检测,影像学检测只能发现胎儿出现出生缺陷以后的影像结果,难以实现早期诊断的目的;而各种针对出生缺陷的生化检测还没有寻找到一种针对某种产前出生缺陷特异性的生物标志物或代谢物谱群,这给出生缺陷临床的诊断的进步带来了困难。代谢组学是继基因组学、转录组学、蛋白质组学后出现的以定量描述生物体内代谢物多参数变化为目标的新兴“组学”,是系统生物学的重要组成部分。代谢组学是关于生物体系(从细胞到整体生物)受刺激或扰动前后(如将某个特定的基因变异或环境变化后)其代谢产物种类、数量及其变化规律的科学。它研究的是生物整体、系统、器官、细胞的代谢物的代谢途径及其所受内在或外在因素的影响。代谢组学主要利用高通量化学分析和多维统计分析为基础的模式识别方法对生物体的扰动进行研究,从代谢产物角度去有针对性地研究疾病的发生、发展及其引发的疾病病理过程的代谢产物并从中寻找共性代谢物以及区别特异性指标(生物标志物),对于疾病及其引发的疾病的病因研究、预防、早期诊断和药物干预评价将可能产生重大影响。金属组学(Metallomics)是继基因组学、转录组学、蛋白质组学和代谢物组学之后提出的一种新的组学。它不仅研究生物体的金属与蛋白结合的化学组成问题,而且为研究金属蛋白和金属酶等生命物质担负生命活动的机理开辟新的途径。目前不能从分子水平上完全了解依赖于金属的生物化学过程,细胞金属成为辅助因子的机理也尚不清楚,人们急需了解金属或类金属在生物体内存在的全部形态信息。弄清金属及其化合物在正常生命过程、重大疾病的发生、诊断和治疗中的作用机理是目前金属组研究的重点和难点。本文建立了基于色谱质谱联用技术的出生缺陷相关疾病的尿液、血清及羊水的代谢组学和金属组学研究方法,考察了妊娠时营养不良的孕鼠和正常孕鼠羊水中代谢物谱和金属微量元素谱的差异;分析了临床诊断孕育神经管缺陷和胚胎停育胎儿孕妇以及正常孕妇尿液和血清代谢物谱和金属微量元素的差异。主要内容如下:1.以不良饮食食谱建立了出生前营养不良的孕鼠模型,利用三甲基硅烷(TMS)衍生,气相色谱质谱联用(GC/MS)以及电感耦合等离子体质谱(ICP/MS)分析研究方法,考察了出生前不良营养条件对孕鼠羊水中小分子代谢物和微量元素的影响,同时结合模式识别及单维统计方法研究了妊娠时营养不良的孕鼠和正常孕鼠羊水中代谢物谱和微量元素谱的差异。主成分分析(PCA)能将正常孕鼠、低蛋白饮食孕鼠和饥荒孕鼠的代谢物谱和微量元素谱区分开来。实验发现出生前不良饮食会使孕鼠羊水中指征蛋氨酸代谢、多巴胺、谷氨酰胺等神经递质代谢通路的生化物质、锌、硒、钴、铷等微量元素发生改变。本研究显示小分子代谢物和微量元素参与了胎鼠的神经发育过程,这些物质在母体中异常可能直接影响胎鼠神经系统的正常发育过程。2.利用氯甲酸乙酯(ECF)和三甲基硅烷(TMS)衍生,气相色谱质谱联用(GC/MS)和气相色谱飞行时间质谱联用(GC/TOFMS)分析方法对正常孕妇、孕育神经管缺陷(NTDs)胎儿孕妇及胚胎停育孕妇的尿液和血清的代谢组进行了研究。结果表明采用正交偏最小二乘法判别分析(OPLS-DA)可以将正常孕妇和胚胎停育孕妇的尿样、正常孕妇、孕育NTDs胎儿孕妇及胚胎停育孕妇血清样本进行较好区分。用单维检验与多维结果进行交互验证,鉴定出多个尿样和血清差异代谢物。这些差异代谢物参与到蛋氨酸循环、同型半胱氨酸代谢、叶酸循环、甲基丙二酸代谢、一碳基团及必需氨基酸代谢这些与NTDs及胚胎停育密切相关的代谢通路。实验结果表明基于气相色谱质谱联用技术的代谢组学技术可以帮助我们区分胎儿出生缺陷孕妇与正常孕妇的代谢物谱,并在小分子代谢物层次帮助上我们认识NTDs和胚胎停育等出生缺陷的生化机制。3.采用超高效液相四级杆串联飞行时间质谱联用仪(UPLC/QTOFMS)技术的代谢组学方法分析了正常孕妇、孕育NTDs胎儿孕妇及胚胎停育孕妇的尿液和血清的代谢组学差异。OPLS-DA模型可以较好的将正常孕妇和NTDs孕妇,正常孕妇和胚胎停育孕妇尿样进行区分。本实验通过液质联用的分析代谢视窗来发现了一些与出生缺陷相关的弱极性和大分子代谢物如叶酸、VitB6和VitB12等,它们在NTDs孕妇血中明显低于正常孕妇,这在气质联用分析的差异代谢物基础之上,为出生缺陷的发病机制的认识提供了更为全面的科学依据。4.本实验以孕育有NTDs胎儿和停育胎儿的孕母及正常孕母的血清为研究样本,采用硝酸湿法消解的ICP/MS分析方法,得到出生缺陷和正常孕母血清中金属(微量)元素的信息,OPLS-DA模型可以将正常孕母和NTDs孕母的血清金属组进行区分。结合对孕母孕周的分析以及已有文献报道,发现NTDs胎儿孕母与正常孕母血清中金属(微量)元素组的差异,这些差异与铅(Pb)、汞(Hg)、铬(Cr)等有毒重金属及微量元素钴(Co)在NTDs胎儿孕母体内的代谢异常密切相关。更多还原

【Abstract】 Birth defects (BDs) are the developmental anomaly statuses before the fetus was born, which including morphological, structural, functional, metabolic and psychoneurotic abnormalities of fetus during the fetal development process. BDs are becoming one of inducements that lead to the death of fetus and disability of child and adults along with the rapid population growth and transformation of disease. In China, there are 51 millions of disabled person and 22 millions of inborn error of metabolism patients and BDs are the main causes of these disabilities, which imposed great financial burden upon our country and society. As multi-factorial and polygenic diseases, BDs have extreme complex etiologies which included not only hereditary defects such as chromosomal and gene abnormality, but also environmental factor based malformation such as poor nutrition, drugs and environmental pollution, and therefore, to find out the BDs related specific biomarkers is the highlight of the diagnosis and therapy of these formidable diseases. To date, it is hard to find out effective prevention and therapy of NTDs except of folic acid supplement during the before and after first trimester of pregnancy. The current prenatal diagnosis methods included imageological results such as transonogram and computed tomography, which are not suitable for most BDs due to these imagings are often late deformity results. There are no specific biomarkers or metabolic profiles in the current clinical biochemistry detection which also limited its usage in BDs diagnosis.Metabonomics or metabolomics is the developing“omics”which quantitative measurement of metabolites, as an integral part of the systems biology encompassing other omics sciences such as genomics, transcriptomics and proteomics. Metabonomics is the quantitative and qualitative measurement of the multiparametric time-related metabolic responses of a complex system to a pathophysiological intervention or genetic modification. The main purpose of metabonomics is to elucidate endogenous metabolic pathway on different biological levels such as system, organ, tissue or cell level. The main characteristics of metabonomics are the high-throughput chemical analysis and multivariate data analysis. Metabonomics based technologies can find out the specific biomarkers or metabolic profiling of complex diseases at nosogenesis, development and prognosis. Metabonomics has become an important highlight for the research of etiology, early diagnosis and drug intervention. Metallomics is another newly developed“omics”science emerging from genomics, transcriptomics, proteomics and metabonomics. The main scopes of metallomics are not only to depict chemical composition of metal and functional protein combination, but also to elucidate the function of metalloprotein and metal enzyme in biosystems. Nowadays, metal based biochemical reaction can not be fully understood on molecular level and the mechanism of metal coenzyme is also a secret. The emphasis and nodus of metallomics is to elucidate the function of metal and its related compound in normal vital movement, etiology and development of complex diseases.In the current dissertation, we tried to establish chromatography in hyphenation with mass spectrometry based metabonomic and metallomic technology to investigate metabolic and trace elemental variation in prenatal malnutrition pregnant rats and pregnant women who were pregnant with BDs fetus and to investigate the applicability of such metabonomics and metallomics method in BDs diagnosis and mechanism research.We used trimethyl silane derivatization, gas chromatography-mass spectrometry (GC/MS) and inductively coupled plasma mass spectrometry (ICP/MS) based metabonomic and metallomic analysis of amniotic fluid (AF) from prenatal malnutrition pregnant rats. PCA scores plot of both GC/MS and ICP/MS data showed similar and unique metabolic signatures of AF in response to the different diets. Rats in the famine group released decreased amounts of methionine, dopa, glutamine, zinc, cobalt, and selenium in the AF. These discriminable variations in the AF may indicate the abnormality of a number of metabolic pathways in fetal rats including the folate cycle and methionine pathway, the monoamine pathway and necessary trace elements metabolism. This study demonstrates the potential of combining profiling of small-molecule metabolites and trace elements to broaden the understanding of biological variations associated with fetal neurodevelopment induced by environmental perturbation.GC/MS and GC/TOFMS based metabonomic approach was used to investigate the BDs related metabolites profiling variation of the urinary and serum samples from pregnant women who were pregnant with neural tube defects (NTDs) and embryo stasimorphy fetus. OPLS-DA model of both GC/MS and GC/TOFMS data showed significant separation tendency between urinary and serum samples on case and control groups. Urine sample from embryo stasimorphy group released increased amounts of methylmalonic acid and decreased amounts of necessary amino acid (MMA), serum sample from NTDs group released decreased amounts of methionine. The urinary and serum metabolites variations in BDs pregnant women may indicate the abnormality of a number of metabolic pathways including methionine cycle, folic acid cycle, homocysteine metabolism, MMA metabolism and TCA. This study demonstrates the potential of metabolic profiling of small-molecule metabolites as a early diagnosis tool to distinguish BDs from healthy control and broaden the understanding of the etiology and mechanism of BDs.UPLC/QTOFMS metabonomic approach was used to investigate the BDs related high molecular weight metabolites variation of the urinary and serum samples from pregnant women who were pregnant with neural tube defects (NTDs) and embryo stasimorphy fetus. OPLS-DA model of UPLC/QTOFMS data showed separation tendency between urinary samples on case and control groups. Serum sample from NTDs group released decreased level of folic acid, vitaminB6 and vitaminB12 as compared with healthy pregnant women.This study broaden the metabolic profiling analytical window in the BDs related diseases.The nitric acid digestion ICP/MS method based metallomics strategy was used to analysis serum trace elements profiling of healthy and BDs pregnant women. OPLS-DA model of ICP/MS data showed separation tendency between NTDs and healthy pregnant women. Serum sample from NTDs group released decreased level of cobalt and increased level of heavy metal such as lead, mercury and chromium as compared with healthy pregnant women. This study demonstrates the potential of trace elements profiling to broaden the understanding of the mechanism of neural tube defects更多还原