人类十二指肠液蛋白质组学研究

发布时间：2018-03-02 02:00

  本文关键词： 十二指肠液 蛋白质组学 标本预处理 蛋白数据库 图谱类型 肿瘤标志物　出处：《中国协和医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 研究背景: 胰腺癌是恶性程度最高的肿瘤之一,据2007年统计结果显示,在美国胰腺癌是肿瘤死因顺位中的第4位。早期发现及早期干预对于胰腺癌患者提高生存率是至关重要的。但遗憾的是,目前临床上应用的肿瘤标志物,其敏感性和特异性都较差。近年来应用蛋白质组学方法寻找肿瘤标志物成为医学基础研究的热点,但目前尚未见到有关于人类十二指肠液蛋白质组学研究的相关报道。 目的与方法: 本课题以人类十二指肠液标本作为研究对象,以1-DE与2-DE进行蛋白分离,以LC MS/MS与MALDI-TOF/TOF进行蛋白鉴定。摸索人类十二指肠液的预处理方法,探索十二指肠液蛋白质组学的研究方法,建立十二指肠液蛋白数据库,并初步探讨十二指肠液替代胰液寻找肿瘤标志物的可行性。 结果: 1)预处理采用超滤离心法,可以达到除盐与蛋白浓缩的目的;以纯水作为超滤置换液,可以保证十二指肠液标本的稳定性;2)行二维凝胶电泳时,十二指肠液一旦与重泡胀液混合,就会出现明显蛋白降解的现象,加入大剂量蛋白酶抑制剂或同时给予更为彻底的蛋白变性处理,有助于减少标本中蛋白的降解;3)通过1-DE对44例患者的标本进行蛋白分离,发现十二指肠液呈现出3种图谱类型,其中1种与胃液图谱相似,怀疑部分标本中有胃液混入;4)通过1-DE对消化系统良性疾病患者的十二指肠液标本进行蛋白分离,以MALDI-TOF/TOF质谱进行鉴定,共鉴定出33种独立的蛋白,绝大部分为胰液蛋白成分,其中有3种为胃蛋白酶类,怀疑部分标本中有胃液混入;5)通过对比发现,十二指肠液与胰液之间从1-DE图谱形态到蛋白鉴定的结果均存在很好的一致性。 结论: 1)超滤置换法是十二指肠液标本预处理过程中一种有效的除盐和浓缩蛋白的方法;超滤置换液选用纯水可保证标本的稳定性;2)行2-DE时,十二指肠液标本中蛋白的降解问题出现在一向等点聚焦过程中,其原因可能是由于重泡胀液提供的碱性环境,使得某些酶类或小分子物质得以激活;3)对十二指肠液1-DE图谱进行了分类与分析,共发现3种图谱类型,其中的Ⅱ型图谱可能是由于十二指肠液标本中混入了胃液;4)建立了消化系统良性疾病患者的十二指肠液蛋白数据库;5)十二指肠液可以起到辅助胰液蛋白质组学研究的作用。
[Abstract]:Background:. Pancreatic cancer is one of the most malignant tumors, according to statistics in 2007, Pancreatic cancer is the fourth leading cause of death in the United States. Early detection and early intervention are essential to improve survival rates in patients with pancreatic cancer. In recent years, the application of proteomics to search for tumor markers has become a hot topic in basic medical research, but there are no reports on proteomics of human duodenal fluid. Objective and methods:. In this paper, human duodenal fluid samples were used as research objects, 1-DE and 2-DE were used for protein separation, LC MS/MS and MALDI-TOF/TOF were used for protein identification, and the pretreatment method of human duodenal fluid was explored. To explore the method of proteomics of duodenal fluid, to establish the database of duodenal fluid protein, and to explore the feasibility of using duodenal fluid instead of pancreatic juice to search for tumor markers. Results:. 1) pretreatment with ultrafiltration centrifugation can achieve the purpose of desalination and protein concentration, and using pure water as ultrafiltration replacement solution can ensure the stability of duodenal fluid. Once the duodenal fluid is mixed with the swelling fluid, there will be obvious protein degradation, which will be treated with a large dose of protease inhibitor or more thorough protein denaturation. Protein separation from 44 patients by 1-DE showed that duodenal fluid showed three patterns, one of which was similar to that of gastric juice. The duodenal fluid of patients with benign diseases of digestive system was separated by 1-DE and identified by MALDI-TOF/TOF mass spectrometry. A total of 33 independent proteins were identified, most of which were pancreatic protein components. Three of them were pepsin, which was suspected to be mixed with gastric juice in some specimens. By comparison, it was found that there was good agreement between duodenal fluid and pancreatic juice from 1-DE map morphology to protein identification. Conclusion:. 1) Ultrafiltration replacement is an effective method of desalination and protein concentration in the pretreatment of duodenal fluid samples, and the ultrafiltration replacement solution can ensure the stability of the samples by using pure water. The degradation of proteins in duodenal fluid appears in the process of isopoint focusing, which may be due to the alkaline environment provided by refrosting fluid. The 1-DE map of duodenal fluid was classified and analyzed. The type 鈪，

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