论文标题:乙肝病毒相关性肝硬化及原发性肝癌血清证候蛋白质组初步研究
Study on Dialectically Assiociated Proteins of Posthepatitic Cirrhosis and Hepatic Cellular Cancer by Serum Proteomics Analysis
论文作者
论文导师 吕志平,论文学位 博士,论文专业 中西医结合临床
论文单位 第一军医大学,点击次数 149,论文页数 88页File Size4564K
2007-04-01论文网 http://www.lw23.com/lunwen_2139522/
Posthepatitic cirrhosis;; hepatic cellular cancer;; syndrome factor analysis;; serum proteomics;; protein gel electrophoresis
研究背景 肝硬化(Hepatic cirrhosis，HC)是许多慢性肝脏疾病的晚期阶段，是肝脏慢性、弥漫性、进行性的病变。引起肝硬化的病因很多，在我国以乙型肝炎病毒感染最为常见。原发性肝癌(primary hepatic Carcinoma，PHC)是常见肿瘤之一，其中90％为肝细胞癌(hepatic celluar cancer，HCC)，其致病原因尚未完全明确，肝炎病毒的感染被认为是原发性肝癌的主要病因，我国大陆地区主要为乙型肝炎病毒感染。慢性病毒性肝炎、肝炎后肝硬化及原发性肝癌三者之间存在着密切联系，从一定意义上已被认为是一个疾病的不同发展阶段。对于HC和HCC辨证论治的临床与实验研究是中医肝病领域中的热点，已有的研究从分子生物、免疫、组织病理、影像学等多角度、多层次入手，初步证实了各证型与相关指标之间的联系。目前为止，还没有较为全面和公认的反映“证”本质的指标出现，仍需要做进一步的研究。 中医的“证”是生命物质在疾病过程中具有时相性的本质的反映，“候”是病变的临床表现。由于中医学对于疾病的认识侧重于整体、宏观、司外揣内，重视疾病某阶段机体的整体状态，辨证论治也即是通过对疾病表现在外的征象，进而推测演绎疾病的病因、病性、病位，由此归纳出证的概念。证候蛋白质组学是指在证候理论指导下，运用功能蛋白质组学的方法，通过探讨证候，特别是同病异证或异病同证的蛋白质差异表达及翻译后的修饰情况，揭示与某一证候形成相关的所有蛋白质及其特征，在整体蛋白质表达的水平上阐明证候的本质。蛋白质是生命活动中多项功能的执行体，实际上每一种生命运动形式，都是特定蛋白质群体在不同时间和空间出现，并发挥功能的不同组合的结果。从组成人体的物质分子方面分析引起“证”的物质基础时，中医“证”的本质是一类具有信使分子性能的蛋白质和肽类分子。蛋白质组研究从整体水平上反应了疾病过程中蛋白质表达的动态演变过程，这与中医辨证论治的认识方法具有极大的相似性。 研究目的 本研究辨病与辨证相结合，从临床密切相关的两种疾病入手，运用蛋白质组技术，分别研究和求证每种疾病特异性蛋白以及二者在相应证候相关性的蛋白质基础。通过对正常和病变样本、异病同证、同病异证之间的蛋白质组成分差异的比较，从整体上发现某些疾病或证候的特有蛋白质的表达，在复杂的多种致病因素作用下寻找证候的共同本质，探讨蛋白质多态性与疾病中医分型、辨证用药之间的系统的有规律的联系。 研究方法 本课题以乙肝相关性肝硬化和原发性肝癌患者为研究对象，在中医理论的指导下，筛选出两种疾病中肝气郁结和肝肾阴虚型病例。运用蛋白质组学研究中常用的二维凝胶电泳(2-DE)分离方法，结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术，分析正常和病变样本、异病同证、同病异证之间的差异蛋白质组成分。 1、原发性肝癌中医辨证分型标准的临床研究 本次辨证标准的研究通过文献调研制定症候调查表、临床辨证资料的收集、因子分析以及临床再检验等四各步骤，初步完成了在一定范围内部分人群HCC辨证分型标准的制定，所制定的辨证标准在临床运用中也取得了较为满意的效果。 2、血清蛋白质组的2-DE分离 由于血清蛋白质成分复杂，各组分间浓度差异大等特点，对血清蛋白质组2-DE分离中的各项技术条件加以优化处理。采用丙酮沉淀与“盐桥”相结合的方法除盐，促进2-DE分离中的有效聚焦；以更长的窄范围胶条提高上样量，增强低丰度蛋白的分离效果；以高伏小时数改善电泳时一向的聚焦效果。通过上述的各项技术改良，完善和提高了原有的技术平台，建立了更为稳定的2-DE分离技术系统。实验中尝试使用试剂盒去除血清中高丰度的白蛋白和IgG，但经试剂盒处理过的血清2-DE分离后蛋白斑点明显减少，蛋白丢失较多，故在实验中未选用该方法。 3、凝胶染色和图像分析 对2-DE分离所获得的凝胶采用新型胶体考染技术，在提高凝胶染色灵敏度的同时，减少对后期质谱检验的干扰。染色和脱色后的PAGE胶以透射扫描仪扫描成像。运用PDQuest-7．1．0软件分析凝胶图像，获取每一个蛋白斑点的表达量、等电点等信息，设定表达量差异2倍以上的为差异表达蛋白，分析结果由软件自动生成，人工加以验证。 4、差异蛋白质斑点质谱鉴定和数据库确认 差异蛋白质斑点进行肽指纹(peptide mass fingerprint，PMF)检测香港大学基因研究中心检测鉴定。根据NCBInr和SWISS-PROT数据库检索分析，寻找与目的PMF相匹配的蛋白质，并检索该蛋白的相关信息。 研究结果 1、通过对临床流调所得资料的因子分析，提取了4个与临床证型契合度较高的公因子。根据统计学分析结果，结合临床验证，将HCC分为肝郁气滞、湿热内蕴、肝肾阴虚、脾肾阳虚、肝血瘀阻5个证型，并制定出相应的辨证标准。 2、获得了较为满意的血清蛋白质组2-DE凝胶电泳图像。改良后的血清2-DE分离技术，即使在不去除高丰度蛋白的条件下也能达到较理想的蛋白分离效果，避免了去除高丰度蛋白所带来的蛋白丢失。 3、经2-DE分离、凝胶图像的软件分析，筛选出具有明显表达差异的蛋白质斑点35个，分布在不同的比较组中。 4、经过对差异蛋白斑点的PMF分析和数据库检测，鉴定出9种差异蛋白质，分别是：载脂蛋A-1、转甲状腺素蛋白、a_1-抗胰蛋白酶、维生素D结合蛋白、触珠蛋白、血清类粘蛋白、转铁蛋白、纤维蛋白原、血清白蛋白前体。 结论 本研究首次运用证候蛋白质组学的方法，在乙肝后肝硬化和原发性肝癌患者中寻找同病异证和异病同证的蛋白质组基础。通过临床研究制定原发性肝癌的辨证标准，采用2-DE联合质谱鉴定技术，对乙肝后肝硬化和原发性肝癌以及其中肝气郁结型和肝肾阴虚型患者的血清蛋白质组进行分析，分离并鉴定出相关差异表达的蛋白质。这些蛋白涉及机体代谢、免疫、凝血、内分泌以及肿瘤细胞增殖代谢等多个领域。通过本次研究发现认为：一、“异病同证”的诊断确实存在蛋白质基础；二、对证本质的研究要坚持“病”与“证”相结合的原则，重视病对证的影响；三、“同病异证”的比较研究是“证”的特异性客观指标筛选的不可或缺的环节。
Background: Hepatic cirrhosis (HC) is the advanced stage of many chronic hepar diseases. It is a chronic, diffused and progressive pathological change in liver. Many factors including virus, bacterium, infection by Schistosoma, chronic alcoholism, drugs and bane damage can cause HC. Hepatitis B infection is the most common cause in our country.Hepatoma is a kind of common tumor and hepatic cellular cancer (HCC) accounts for about 90%. Pathogeny of HCC has not been clearly known yet. Hepatitis virus infection which is mainly caused by hepatitis B in mainland of China is considered to be the major cause. There are close relationships among chronic viral hepatitis, posthepatitic cirrhosis and HCC. In some extent, they are considered three different developing phases of the same disease. The determinations of treatment based in pathogenesis obtained through differentiation of symptoms and signs of HC and HCC are hot spots of traditional Chinese medicine. The clinical and experimental studies of determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs of HC and HCC are hot spot in traditional Chinese medicine hepatopathy. The relationships between each pattern of syndrome and its related indices have been tentatively confirmed by multi-perspective and multistrata research of molecular biology, immunology, histopathology and imageology, et al. So far, there has not an widely and generally accepted index which can show the nature of Zheng. There needs further studies. Traditional Chinese medicine puts emphasis on the whole status of bodies at every phase of the disease. Determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs means deducing the causes of a disease, nature of disease and location of disease according to the outer signs of a disease. By approaching syndrom, especially the heterologous proteins expression and modified status of different diseases with same symptome and among different symptome with same disease, syndrom Proteomics guided by the theory of syndrom,applying the method of functional proteome to reveal the all proteins and their character related to the formation of syndrom and to interpret the nature of syndrom in the level of integer proteins expression. Proteins can perform all kinds of functions in vital movement. In fact, every kind of life activities is a kind of movement. They are the combined result of specified proteins appearing in different time or space.When we consider the matter basis which causes the syndrom, the nature of syndrom is a kind of proteins or peptides with the function of messenger molecule. Proteomics research reflects the dynamic evolution process of proteins expression of disease in integrated level. It is extremely similar to the method of determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs in traditional Chinese medicine. Objective: Combining differential diagnosis of diseases and differentiation of symptoms and signs, beginning with two clinically correlative diseases and using proteome techniques, to research and prove the heterologous proteins of each disease and the protein bases of dependability in corresponding syndrom of two diseases. To discover the expression of some heterologous proteins in some diseases or syndrom to find the common nature of syndrom in all kinds of complex etiological factors, to approach the systematic and regular relationship among protein polymorphism, disease grouping and medication in traditional Chinese medicine by comparing the differences of protein ingredients between normal samples and sick, among different diseases with same symptome and among different symptome with same disease. Methods: Patients with type B hepatitis correlated HC and patients with HCC are served as research targets. Guided by traditional Chinese medicine theory, the patients with depression of liver-QI and hepatic and renal yin deficiency are screened out. Applying the commonly used 2D electrophoresis in proteomics and combining MALDI-TOF-MS techniques, the different protein components between normal samples and sick, among different diseases with same symptome and among different symptome with same disease are analyzed. 1. Establishing the standard of differentiation of symptoms and signs for classification of syndrome.By four steps of symptom questionnaire design, clinical information collection, factor analysis and clinic retest, this study formulates the standard of differentiation of symptoms and signs for classification of syndrome scientifically and systematically in some range of HCC patients. The standard has achieved pleasant effect in clinic. 2. 2-DE separating for serum proteome.The specification in the 2-DE separation of serum proteins has been optimized because of the complicated components of serum proteins and their big differences in concentration. Using both methods of acetone precipitating and salt bridge for dechlorination, 2-DE separation becomes more effective. Increasing the sample amount applying longer narrow-range adhesive tapes, the separation effect of low abundance proteins becomes better. The focusing results of time-phase in electrophoresis have been improved time of 8000V.Using these improved techniques, the original technique platform has been improved and 2-DE separation system become more stable. We tried to remove the abundant albumin and IgG in serum using kit. However, when treated with the kit, the protein speckle separated by 2-DE decreases significantly and a lot of proteins have been lost. Thus, we did not apply this method to our study. 3. Gel dyeing and image analyzing.The gel were dyed with "sliver blue". The gel dyeing sensitivity is elevated. At the same time, the interference cause by MS test at later period decreases. We get images of PAGE gel dyed and decolored after transmission scanning. PDQuest-7.1.0 is operated to analyze the gel images in order to get information of expression amount and isoelectric point. Protein which have two times expression amount than others is set to be a heterologous protein. The analyzing results are generated by software then verified by handwork. 4. Identifying the heterologous protein speckles using MS and database confirming The peptide mass fingerprint (PMF) test of the heterologous protein speckles is carried out by Gene Research Center of Hong Kong University. By scanning and analyzing the database of National Center for Biotechnology Information, we want to find the proteins which match the PMF and some information about these proteins. Results: 1. By analyzing the materials acquired from clinic, four factors which are finely agreeable to clinical pattern of syndrome are extracted. According to the statistics analysis together with clinical check, HCC is classified to five pattern of syndrome. There are stagnationof QI due to depression of the liver, endoretention of damp heat, hepatic and renal yin deficiency, asdthenic splenonephro-yang, blood stasis of hepatic blood. Correlative standards formulate. 2. High quality serum proteome 2-DE images have acquired. Applying the reformed serum 2-DE separating techniques, an ideal separation effect has achieved and protein loss is avoided even if the high abundance proteins are not removed. 3. 35 protein speckles which have obvious expressing differences have been screened out after 2-DE separation and gel image analysis. They are distributed in different groups. The exact distribution is shown in the second section of the experiment part. 4. Analyzed by PMF and detected in the database, 9 difference proteins are identified. They are Apo A-1, T4, a_1-antitrypsin, Vitamin D binding protein, haptoglobin, acid glycoprotein, transferring, metaglobulin, albumin mutant R218p and serum amyloid protein precursor. The exact distribution of each group is shown in the second section of the experiment part. Conclusion: In this study, we first use the syndrom proteomics methods to find the proteome basis of different diseases with same symptome and different symptome with same disease. By establishing the differentiation standard of symptoms and signs according to clinical research, applying 2-DE combined mass identifying technique, analyzing serum proteome of patients with depression of liver-QI and hepatic and renal yin deficiency in HC and HCC, the relative heterologous proteins are separated and identified. These proteins are involved in many kinds of human body activites such as metabolism, immunization, blood clotting, endocrine and cancer cell proliferation, and so on. We discover in this study that: 1. To study the nature of Zheng, the principal of combining disease with syndrome should be insisted on. Because different etiological factors may cause the different protein expression in the same pattern of syndrome of different diseases; 2. "Different diseases with same syndrome" the diagnosis truly has the proteinfoundation. 3. Research technique of "Different syndromes with same disease" is the way to flit the specific object target of syndrome nature.
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