【作者】 米薇；

【导师】 钱小红； 应万涛；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药物分析， 2008， 博士

【摘要】 肝脏是人体最重要的器官之一,复杂程度仅次于脑,它是多种物质进行代谢的消化器官,并执行其它多种功能。肝脏疾病影响着超过全世界10%的人口,肝脏疾病的治疗也仍然困难重重,原因是缺乏有效诊断,分级和预后方法。由国际人类蛋白质组组织发起的人类肝脏蛋白质组计划已于2003年正式启动。肝脏蛋白质组的研究是一项系统而复杂的工程。首先肝脏具有非常复杂的蛋白质组成,如何将这些蛋白质分离鉴定本身就是一个挑战;其次,蛋白质组学研究的目的并不是简单的呈现蛋白质本身,而是要研究这些蛋白质在肝脏这一复杂系统中的功能,动态、整体、定量地考察肝脏疾病发生发展过程中蛋白质的变化,寻找疾病诊断的特异标志物和药物治疗的靶标。因此,肝脏蛋白质组的研究向蛋白质的分离鉴定技术、亚细胞蛋白分离和富集技术以及定量蛋白质组技术提出了很高的要求。本论文研究的目的是利用蛋白质组技术,一方面对分离鉴定技术进行优化,构建适合肝脏这种复杂蛋白样品蛋白质组研究的技术平台,尽可能的分析肝脏的全蛋白质组,从全局把握肝脏这一人体重要器官的生理功能,也为肝脏疾病研究提供参考数据;另一方面开发新的分离和富集技术,分析更高纯度的细胞器蛋白、在生命过程中起重要作用的翻译后修饰蛋白、低丰度蛋白和高疏水性蛋白,为全面、深刻的理解肝癌转移这一复杂过程提供信息依据,寻找有潜力成为早期诊断肝癌、预测转移的生物标记和干预治疗的靶分子。另外建立和优化定量蛋白质组技术,研究肝癌细胞以及肝疾病组织的差异表达,寻找与病理改变有关的蛋白质和疾病特异性蛋白质。本论文由4个部分组成。第一章概述了蛋白质组学的技术进展,包括以凝胶质谱联用技术、多维色谱质谱联用技术为主的分离鉴定技术,对膜蛋白以及糖蛋白这些丰度低、功能重要蛋白的富集技术,以及用于研究差异蛋白质的定量蛋白质组技术。在此基础上提出了本课题的研究内容。第二章阐述基于双向凝胶电泳与质谱联用技术研究正常人肝脏全蛋白的表达谱研究。大规模的蛋白质组研究需要高分辨蛋白质分离技术和高通量、高灵敏度的鉴定技术。在双向凝胶电泳第一向等电聚焦水平上,我们从上样方式、水化液配方、聚焦时间等方面优化了碱性蛋白的分离条件,利用超放大胶分离技术搭建了高分辨的双向凝胶电泳分离平台,构建了人类肝脏蛋白质2-DE参考谱,检测到5481个蛋白质点。这是目前国际上最为全面的人体器官蛋白质组2-DE参考谱,为其它肝病的研究建立了良好的参照系。我们成功鉴定了429个非冗余蛋白质,对pH4-7、pH5-6、pH5.5-6.7、pH6-9部分蛋白质点在胶上进行了注释,由此构建了人肝2-DE蛋白质表达谱数据库。对蛋白质的理化性质、功能及亚细胞定位进行了全面分析。90%的蛋白分子量都分布于10KDa-80KDa的范围,pI分布呈典型的双峰形式。大部分蛋白GRAVY值小于0,说明鉴定到的蛋白主要是亲水性蛋白。鉴定的大部分蛋白质具有催化活性,参与代谢活动,其他蛋白与基因、蛋白质表达与降解,细胞骨架形成与离子传输,信号转导,细胞生长与调控等功能相关,充分体现了肝脏所承担的生理功能。肝脏的亚细胞器修饰谱研究是在全蛋白表达谱基础上对肝脏蛋白质组的进一步深入研究。第三章所述工作是建立一种利用生物素酰肼标记和亲和素纯化富集糖蛋白和糖肽的方法,并将此方法用于HepG2肝癌细胞膜糖蛋白研究。首先对标准蛋白的生物素标记-亲和素纯化进行了一系列的条件考察,结果证明该方法能特异性的富集糖蛋白,并且采用SDS buffer煮沸条件能将富集蛋白从链霉亲和素上完全洗脱。我们设计了三条技术路线,从糖蛋白和糖基化位点质谱鉴定的结果,考察三条技术路线的富集效率和富集特异性。结果显示从肽段水平上富集生物素化糖肽,操作简单,富集效率和特异性高,更适合复杂样本的分析。采用建立的生物素酰肼-亲和素纯化法分别从蛋白水平和肽段水平对HepG2肝癌细胞膜糖蛋白进行了富集研究。蛋白水平富集的膜糖蛋白共鉴定到171个跨膜蛋白,占鉴定蛋白的31%,其中93(54%)个蛋白具有2个或2个以上的跨膜区,44个是跨膜糖蛋白。肽段水平富集的膜糖肽鉴定到70个非冗余糖肽,48个糖蛋白,73个糖基化位点。其中发现了很多肝癌及肝癌转移相关蛋白,涉及了肝癌转移的多个过程,如代谢,细胞运动和侵袭,信号转导等,为全面、深刻的理解肝癌转移这一复杂过程提供了丰富的数据。本工作首次构建了HepG2肝癌细胞膜糖蛋白数据,该数据库对肝癌转移复发相关研究具有一定价值。为了更好的理解肝癌的发生机理和寻找阳性率高、特异性强的肝癌早期诊断标志物,第四章中我们应用基于细胞培养稳定同位素标记(SILAC),液相色谱-傅立叶变换离子回旋共振质谱和定量分析软件MSQuant联用的定量蛋白质组策略进行了差异蛋白质分析。应用精氨酸同位素标记,对AFP阳性肝癌细胞系HepG2和AFP阴性肝癌细胞系SK-HEP-1以及正常肝细胞系HL-7702进行了差异比较研究,发现了一些与肝癌密切相关的蛋白,其中蛋白分子TGM2在AFP阴性和阳性肝癌细胞中有显著差异,有可能成为肝癌诊断的标志物。更多还原

【Abstract】 Human liver is the largest organ in the body, probably second only to the brain in complexity. It takes on many important physiological functions including the main digestive function for the metabolism of most substances.Liver diseases are great challenges for modern medicine with extremely poor prognosis due to failure of effective early diagnosis and stage analysis. In order to address this serious health issue, the human liver proteome project was initiated by HUPO in 2003.The research of liver proteome is a systematic and complicated project. Firstly, it was absolutely a challenge to separate and identify all liver proteins because of the very complex protein compositions. Secondly, the goal of liver proteome research is not just to identify all expressed proteins in human livers, but also to study the function of them. Recent advancement in proteomics allow us to study global patterns of protein content and activity and how these change during development or in response to disease, it has boosted our understanding of systems-level and mechanism of disease. In addition, proteomics benefits the identification of new drug targets and development of new diagnostic markers in clinical research. Therefore, the development of new technologies will play a key role in proteome research.Tremendous progress has been made in proteomics during the past few years. With the notable technology developments, one of the aims of our research is to optimize separation and identification techniques and develop an integrated analysis platform which can separate, detect, and identify as many liver proteins as possible. Then, the studies of new sample preparation technologies should be strengthened, and the targets of these studies should be concentrated on the enrichment low-abundance proteins and elucidation of the post-translational modification protein profiles. At last, we wanted to establish a quantitative strategy to analyze the differentially expressed protein profiles of human hepatocellular carcinoma (HCC), which might be helpful to identify new biomarkers of HCC and improve early detection of HCC for the patientsThis dissertation consists of 4 parts and contents are summarized as follows:In the first chapter, the status quo of proteomics and the technology developments in proteomics such as 2-DE, MUDPIT, enrichment of membrane proteins and glycoproteins, and quantitative methodologies, were summarized.The goal of the study in chapter two was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis and make a reference map of human normal liver proteins for the comprehensive analysis of human liver proteome and the other related research. To tackle the poor resolution in the alkaline pH range of 2-DE gels, we have optimized isoelectric focusing protocol including sample application using cup-loading at the anode and modification of rehydration buffer. With the application of optimized protocol we get reproducible better resolution both in analytical and preparative 2-DE gels. Narrow pH range ultra-zoom 2-DE gels were developed and optimized to improve the resolution and enhance the detection of low abundance proteins. High resolution patterns of human liver in pH gradients 4.5-5.5, 5-6, 5.5-6.7, 6-9 were presented. The ultra-zoom gels pH4.5-9 revealed a total of 5481 protein spots.429 unique proteins were successfully identified, and some of which were labeled on the 2-DE maps and annotated by GO analysis. It is hoped that the visualized reference map of human liver proteins presented in this work will be valuable for comparative proteomics of liver disease.Based on the expression profile of human liver proteome, the research of subcellular organelles and post-translational modification was the further study of human liver proteomics. In chapter three, we first developed a purification strategy to effectively enrich membrane glycoproteins, which involves biotinylation of cell surface membrane glycoproteins and affinity enrichment of the membrane glycoproteins.To address the practicability of the method, the whole strategy was first employed to isolate and identify the glycoproteins from a standard protein mixture. The result showed that the glycoproteins were unambiguously enriched and identified. Three technique lines were compared to investigate the efficiency and specificity of enrichment. It proved that capturing glycosylated peptides can effectively reduce sample complexity and fit for complexity. Then, we used the method to analyze hepatocarcinoma cells’ membrane glycoprotein for the potential biomarker discover for liver cancer. From protein level, 171 integral membrane proteins were identified and 54% had more than one TMD, of which 44 were integral membrane glycoproteins. Frome peptide level, 70 unique glycopeptides with 73 glycosylation sites were identified, and it resulted in the identification of 48 membrane glycoproteins. Therefore, we have build up a HepG2 membrane glycoprotein database in the first time, and this data should have a great value for analysis of HCC metastasis.The work in chapter four is focus on the differentiated expressed protein profiles of hepatocellular carcinoma cell lines. To improve early diagnosis of HCC as well as better understanding the mechanisms underlying tumorigenesis, A quantitative proteomic analysis approach, stable isotope labeling with amino acids in cell culture (SILAC) combined with LTQ-FT-MS/MS identification and MSQuant quantitative software, was used to explore differentially expressed protein profiles between normal (HL-7702) and cancer (HepG2 and SK-HEP-1) cells. Some HCC related proteins were found out, and it suggested that TGM2 may serve as a novel candidate involved in HCC. These novel findings may add important clues to identify new biomarkers of HCC and improve early detection of HCC.更多还原