【作者】 赵海丰；

【导师】 何成彦；

【作者基本信息】 吉林大学 ， 病理生理学， 2010， 博士

【摘要】 大肠癌在不同地区、种族、年龄发病率不同。在西方国家发病率居第二位,并且发病率和死亡率均有逐年上升的趋势。大肠癌发病的年龄趋向老年化,根据收集分析我国20世纪70年代文献报告,我国大肠癌患者的中位年龄在45岁左右,比欧洲的报告提前12-18年。我国70年代文献中40岁以下大肠癌病人一般占35%左右,30岁以下病人占12%左右。而近5年的数据表明大肠癌中位发病年龄为57岁左右。大肠癌发病中位年龄后移,发病进一步向高年龄组集中。因此,高年龄组无疑是大肠癌发病的高危人群。老年人大肠癌如今已经成为国际上医学研究的热点之一。很有必要对其发病、转移、复发机制进行研究。传统上,国内外学者对大肠癌研究主要集中在:①流行病学调查,前瞻性;②回顾性比较研究;③尸体解剖分析。随着科学进步,对大肠癌研究的方法愈来愈多,层面越来越高。如组织化学染色、PCR方法、蛋白水平研究等等。近年蛋白质水平研究的迅猛发展,为大肠癌的研究提供了又一新的方法。本实验通过双向凝胶电泳与质谱技术和二维液质联用技术及免疫印迹技术,鉴定出临床病理分期中大肠癌DukesB期肿瘤和癌旁组织的差异蛋白,为大肠癌临床诊断及发病机制的研究寻找蛋白标志物;并可进一步为大肠癌的早期诊断、生物学治疗等寻找新的靶点。实验结果:应用双向凝胶电泳与质谱技术实验最后得到大肠癌和癌旁组织差异蛋白21个,其中有14个肿瘤组织蛋白质表达上调,7个肿瘤组织蛋白质表达下调。应用二维液质联用技术分析最后得到大肠癌和癌旁组织差异蛋白44个,其中有21个肿瘤组织蛋白质表达上调,23个肿瘤组织蛋白质表达下调。两种技术路线所得实验结果中共有13个蛋白得到一致的鉴定结果,其中:在肿瘤组织中表达上调的有7个蛋白质,如Hemoglobin Lepore-Baltimore,60 kDa heat shock protein,ATP synthase subunit alpha,Keratin type I cytoskeletal 18,Protein disulfide-isomerase A3,Alpha-enolase,Isoform 1 of Nucleophosmin;在肿瘤组织中表达下调的有6个蛋白质,如Isoform 1 of Desmuslin,Transgelin,Desmin,Calponin-1,Heat shock protein beta-1,Isoform 2 of Vinculin。更多还原

【Abstract】 The Colorectal cancer is one of current global most common mali-gnant tumors, its incidence rate and the mortality rate is increasing year by year. The Colorectal cancer tended to shift and recur easily, and has become the5th in mortality rate of maligrant tumor in our country, it is quite necessary to conduct the research of its morbidity, the shift and the recrudescence mechanism .Traditionally, the domestic and foreign scholars,researches of colorectal cancer mainly focus on :①Epidemiology investigation, foresightedness;②Foresighted and retrospective comparative studies③autopsy analysis. Nowadays, along with scientific progress, the methods for the study of colon cancer are increasing rapidly, and the studying fields are more and more broad. Such as histochemistry dyes, PCR method, protein studies and so on. Particularly in recent years, the research in Protein levels developed more quickly,and the study on colorectal cancer has steped into a new world.With the human genome project’s implementation and advancement,life science research has entered the post-genome era. In this era, the main studying object of life science is functional genomics, including structural genomics and proteomics research.Although there are many species’ genome has been sequenced, but more than half of gene’s function is unknown in these genomes . Currently, the strategies of the functional genomics uses, such as gene chip, serial analysis of gene expression (SAGE), which are considered in the context of mRNA from the cells , and its premise is that the level of mRNA of cells can reflect the protein expression levels. But this is not entirely the case, in the mRNA point of view, actually, it only includes the level of transcription regulation, and can not fully represent the level of protein expression. Protein’s complex post-translational modification, protein’s sub-cellular localization or migration, and protein-protein interactions is almost impossible to judge from the mRNA level.Needless to say, protein is executor of physiological functions, is the direct embodiment of the phenomenon of life, and the study of protein structure and function will directly clarify the conditions of life under physiological or pathological changes in the mechanism. The existence forms and activity patterns of the protein itself, such as post-translational modification, protein-protein interactions and protein conformation and so on, still depends on the direct study of proteins to resolve. Although the protein’s special nature, such as variability, diversity and so on, has led to the result that the research techniques of protein are far more complex and difficult than nucleic acid technology , but it is these characteristics affect the entire life course.The traditional study approach of a single protein has been unable to meet the requirements of post-genome era, it is because [2]:①Biological phenomena’s occurrence is often multi-factors,it will inevitably involve multiple proteins;②the involvement of multiple proteins is interwoven into a network, or parallel to occur, or showed a cascade relationship;③In the implementation of physiological functions, the performance of proteins are diverse, dynamic, it is not as basically fixed as the genome. Therefore,if one need to have a comprehensive and in-depth understanding of the complexity of life, it is essential to study protein on a overall, dynamic, network level. Thus, in 1994, Australia’s Macquarie University, Wilkins and Williams first proposed proteome concept, which originated from the heterozygosity of two words,protein and genome , which is defined as: all types of proteins expressed by a cell in a particular physiological or pathological state. Proteomics which takes all proteins expressed by the genome, namely protein group,as studying object, is a new area of research. Proteomics is divided into two types: 1、expressional proteomics which is to study the change of quantity of protein expression between different samples .2、functional proteomics which establish cell signal transduction pathway network through the separation of protein complexes and the systematic study protein-protein interactions . Proteomics has a rich studying content ,a great many technology platform and more and more advanced technological means. In that case, rational selection of clinical specimens, is the key to the expressional proteomics studies.Generally, International organizations still follow the Dukes improved TNM staging and the staging method proposed by UICC. According to the Dukes law’s supplementin our country , divides into: Cancers limited in the intestinal wall is within the DukesA period. Serosal invasion through the intestinal wall and / or serosa things, but no lymph node metastasis is the B phase. With lymph node metastasis belongs to the C phase, in which lymph node metastasis is limited to the vicinity, such as colon wall and lymph nodes are for the C1 period; if transferred to the Department of Film and mesangial root node is the C2 period. Already have distant metastasis or peritoneal metastasis or extensive invasion to adjacent organs and can not be removed is the D periodBased on the above principle, this experiment identified stages of clinical pathology, the Patient tumor organization of Colorectal cancer in Dukes B period and different proteins of adjacent tissues in the pathological classification through the proteomic approach.,it can provide protein markers for the pathological classification of colorectal cancer ;it can be further used to find new targets for early diagnosis and biology treatment of colorectal cancer .Under the application of two-dimensional gel electrophoresis and mass spectrometry experiments , we finally obtained organization different proteins of tumors and adjacent tissues 21, including 14 up- regulated proteins and 7 protein down. With the application of two-dimensional LC-MS analysis, we could obtain 44 organization different proteins of cancer and adjacent tissues, including 21 up- regulated proteins and 23 protein down. From the two technical route we find 13 proteins to obtain the consistent appraisal result, and including 7 up- regulated proteins ,for example, Hemoglobin Lepore-Baltimore, 60 kDa heat shock protein, ATP synthase subunit alpha , Keratin type I cytoskeletal 18 , Protein disulfide-isomerase A3,Alpha-enolase,Isoform 1 of Nucleophosmin;and 6 protein down, for example, Isoform 1 of Desmuslin,Transgelin,Desmin,Calponin-1,Heat shock protein beta-1,Isoform 2 of Vinculin.In order to confirm the accuracy of our experiment,we selected two protein(Enolase,Hemogobin) among the 21different proteins of cancer and adjacent tissues by Dimensional gel electrophoresis and mass spectrometry experiments respectively and verified the accuracy of two-dimensional gel electrophoresis by immunity signature experiment; in the same way , we also selected two protein(Vinculin,Talin)among 44 different proteins which were finally received from tumor and adjacentissues in the two-dimensional liquid chromatography analysis and verified the accuracy of Two-dimensional LC-MS experiment by immunity signature experiment. In addition ,we discussed the four proteins above ,which further illustrates their meaning in the incidence of disease ,colorectal cancer diagnosis or treatment and other aspects.更多还原