【作者】 李俊材；

【导师】 傅仲学；

【作者基本信息】 重庆医科大学 ， 外科学， 2008， 硕士

【摘要】 细胞转移是恶性肿瘤的重要特征。肝脏是大肠癌转移最常见的器官。几十年来,关于结肠癌肝转移的临床诊治及基础研究做了大量工作,但其总体预后并未得到显著改善,难以早期诊断而失去手术时机是重要原因之一。血液组分变化可充分反映机体生理、病理过程,容易获取并易于监测,故一直是筛选疾病诊断标志物的最佳研究对象。目前常用的结肠癌肝转移血清标志物尚不能完全满足临床需求,因此,从血清中筛选新的结肠癌肝转移标志物受到医学研究人员和临床医生的高度关注。目前蛋白质组学(Proteomics)已成为后基因时代生命科学研究新的热点,其定义为:研究一个细胞、组织或完整机体在某个特定时空条件下全部蛋白质的表达、功能及其相互作用方式。其特点是以系统生物学的研究思路,采用高分辨率的蛋白分离手段和高通量的蛋白鉴定技术,全景式地研究各种特定情况下的蛋白表达谱。不断发展、完善的蛋白质组学研究策略与技术正成为现代生物医学研究新的强有力工具。从血液中寻找特异性生物标志物用于结肠癌肝转移等诊断是目前临床急需解决的难题,本研究使用差异蛋白质组技术包括双向凝胶电泳(Two-dimensional polyacrylamide gel electrophoresis,2-DE)、基质辅助激光解吸离子化飞行时间质谱( Matrix-assisted laser desorption/ionization mass spectrometry-time of flight, MALDI-TOF)技术对结肠癌肝转移患者和结肠癌患者血清蛋白进行分析,获得了一些可能与结肠癌肝转移相关的标志蛋白质,推测其差异蛋白质的功能,并从蛋白质整体水平上探讨这些差异候选蛋白质是否与结肠癌可能的发生或转移机制相关,并为开展临床早期诊断、寻找治疗靶点等方面提供有价值的理论依据。第一部分结肠癌和结肠癌肝转移患者血清样品的处理目的:纯化血清蛋白质,减少高丰度蛋白对电泳的干扰。方法:参照人类蛋白质组研究组织(Human Proteomo Organization, HUPO)的血清蛋白质组计划(Plasma Protemo Project,PPP)所推荐的方法采集血液标本,利用PPP推荐使用的多种蛋白质亲和清楚系统(Multiple Affinity Removal System,MARS)同时去除血液中的多种高丰度蛋白质,并通过蛋白电泳图谱比较前后的血清蛋白分离情况。结果:使用了蛋白清楚系统去除血清高丰度蛋白后,血清蛋白点数由530个增加到810个,建立了稳定的血清白蛋白和IgG去除方法。结论:多种蛋白质亲和清楚系统能有效去除血清中的高丰度蛋白,减少了高丰度蛋白对其的干扰。第二部分结肠癌肝转移患者血清双向电泳图谱的建立及优化目的:建立并优化血清蛋白质组双向电泳分离技术。方法:对2-DE的各种条件进行调整、优化,通过双向电泳图谱比较各种方法对血清蛋白的分离效果。结果:采用pH4-7胶条,含尿素9M、硫脲2M的上样缓冲液,12.5%的SDS-PAGE胶提高了血清蛋白的分离效果。结论:血液中高丰度蛋白质的去除能提高了2-DE图谱的斑点数和分辨率。成功建立了血清蛋白质的纯化技术,为寻找疾病的血清学标志物的研究奠定基础。第三部分结肠癌肝转移相关蛋白质的鉴定目的:分析结肠癌和结肠癌肝转移血清的蛋白质表达差异。获取两者蛋白表达差异。方法:使用基质辅助激光解吸/电离飞行时间质谱对结肠癌和结肠癌肝转移患者血清的双向电泳图谱中的差异蛋白质点进行鉴定。结果:成功鉴定出TGF-β1、Geranylgeranyl transferase type-1等6个蛋白在结肠癌肝转移组血清中表达上调,Protein kinase C等4个蛋白在对照组血清中表达上调的蛋白质。结论:结果表明在结肠癌肝转移患者和结肠癌患者中存在差异表达蛋白质,利用蛋白质组学这一高通量的筛查技术可望从中发现与结肠癌肝转移相关的标志物。更多还原

【Abstract】 Cell metastasis is the most important character of malignant tumor. The carcinomatous metastasis of colon cancer usually happen on liver. Despite substantial progresses made in colon carcinoma hepatic metastasis clinical and basic research, the overall prognosis of colon carcinoma remains dismal because of the late diagnosis and low resection rate. Although CEA can be an biomarker, the specificity and sensitivity of which are still not satisfied. Therefore, screening novel colon carcinoma hepatic metastasis biomarkers are still needed.Proteomics is becoming the hot field of biological study in the functional genomic era. The scope of proteomics is to study the expression profile, function and cross reactivity of all cell, tissue or organism proteins under specific circumstances. Because of the high throughput and resolving power, proteomics techniques are becoming strong tools for contemporary medical research.The challenge of clinical proteomic studies is to link protein expression profiles to specific disease phenotypes and to find out relevant biomarkers in order to develop diagnostic tools. Two-dimensional electrophoresis (2-DE) is a highly resolving technique for arraying proteins by isoelectric point and molecular mass. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available colon carcinoma hepatic metastasis serum. Protein identification was done by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI -TOF-MS).PART ISAMPLE HANDING OF SERUM OF COLON CARCINOMA HEPATIC METASTASISObjective:To establish and evaluate the method of removing albumin and IgG in serum proteomics by Two-dimensional electrophoresis(2-DE).Methods: The blood samples were collected in accordance with Human Proteome Organization Plasma Proteome Project. After depleting of the six plasma proteins of highest abundance by the MARS, the remaining low- and medium-abundance serum proteins were isolated and concentrated.Results: The method of removing serum albumin and IgG and 2-DE was established stably, increasing sharpness of separation of low-abundance proteins and decreasing the disturbing of high abundance proteins in 2-DE.Conclusion: The Multiple Affinity Removal System can depleting of the six serum proteins of highest abundance.PART IIESTABLISHMENT OF SCREENING SERUM BIOMARKERS BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESISObjective:To establish a two-dimensional polyacrylamide gel electro- phoresis(2-DE)technique for serum proteomics.Methods: Various conditions of serum proteomic 2-DE were adjusted and optimized.Results: A steady 2-DE technique was established.Conclusion: The established method of purifying serum proteins may be foundation in searching serology marker of diseases. PART IIIIDENTIFICATION OF SERUM PROTEINS RELATED TO COLON CARCINOMA HEPATIC METASTASISObjective:To analyze the 2-DE protein separation and identification by PMF in colon carcinoma and colon carcinoma hepatic metastasis.Methods: We used 2-DE and MALDI-TOF based proteomics to compare the protein profiles between colon carcinoma and colon carcinoma hepatic metastasis.Results: 14 protein spots were discovered differently and 10 were identified by PMF,including 6 proteins as TGF-β1、Geranylgeranyl transferase type-1 were up-regulated and 4 proteins as Protein kinase C were down-regulated in colon carcinoma hepatic metastasis.Conclusion: 2-D gel images of colon carcinoma and colon carcinoma hepatic metastasis showed different protein expression. The variety of proteins may be concerned with metastatic mechanism of colon carcinoma hepatic metastasis possibly.更多还原