本文關(guān)鍵詞： 十二指腸液 蛋白質(zhì)組學(xué) 標(biāo)本預(yù)處理 蛋白數(shù)據(jù)庫 圖譜類型 腫瘤標(biāo)志物　出處：《中國協(xié)和醫(yī)科大學(xué)》2008年博士論文　論文類型：學(xué)位論文

【摘要】： 研究背景: 胰腺癌是惡性程度最高的腫瘤之一,據(jù)2007年統(tǒng)計(jì)結(jié)果顯示,在美國胰腺癌是腫瘤死因順位中的第4位。早期發(fā)現(xiàn)及早期干預(yù)對(duì)于胰腺癌患者提高生存率是至關(guān)重要的。但遺憾的是,目前臨床上應(yīng)用的腫瘤標(biāo)志物,其敏感性和特異性都較差。近年來應(yīng)用蛋白質(zhì)組學(xué)方法尋找腫瘤標(biāo)志物成為醫(yī)學(xué)基礎(chǔ)研究的熱點(diǎn),但目前尚未見到有關(guān)于人類十二指腸液蛋白質(zhì)組學(xué)研究的相關(guān)報(bào)道。 目的與方法: 本課題以人類十二指腸液標(biāo)本作為研究對(duì)象,以1-DE與2-DE進(jìn)行蛋白分離,以LC MS/MS與MALDI-TOF/TOF進(jìn)行蛋白鑒定。摸索人類十二指腸液的預(yù)處理方法,探索十二指腸液蛋白質(zhì)組學(xué)的研究方法,建立十二指腸液蛋白數(shù)據(jù)庫,并初步探討十二指腸液替代胰液尋找腫瘤標(biāo)志物的可行性。 結(jié)果: 1)預(yù)處理采用超濾離心法,可以達(dá)到除鹽與蛋白濃縮的目的;以純水作為超濾置換液,可以保證十二指腸液標(biāo)本的穩(wěn)定性;2)行二維凝膠電泳時(shí),十二指腸液一旦與重泡脹液混合,就會(huì)出現(xiàn)明顯蛋白降解的現(xiàn)象,加入大劑量蛋白酶抑制劑或同時(shí)給予更為徹底的蛋白變性處理,有助于減少標(biāo)本中蛋白的降解;3)通過1-DE對(duì)44例患者的標(biāo)本進(jìn)行蛋白分離,發(fā)現(xiàn)十二指腸液呈現(xiàn)出3種圖譜類型,其中1種與胃液圖譜相似,懷疑部分標(biāo)本中有胃液混入;4)通過1-DE對(duì)消化系統(tǒng)良性疾病患者的十二指腸液標(biāo)本進(jìn)行蛋白分離,以MALDI-TOF/TOF質(zhì)譜進(jìn)行鑒定,共鑒定出33種獨(dú)立的蛋白,絕大部分為胰液蛋白成分,其中有3種為胃蛋白酶類,懷疑部分標(biāo)本中有胃液混入;5)通過對(duì)比發(fā)現(xiàn),十二指腸液與胰液之間從1-DE圖譜形態(tài)到蛋白鑒定的結(jié)果均存在很好的一致性。 結(jié)論: 1)超濾置換法是十二指腸液標(biāo)本預(yù)處理過程中一種有效的除鹽和濃縮蛋白的方法;超濾置換液選用純水可保證標(biāo)本的穩(wěn)定性;2)行2-DE時(shí),十二指腸液標(biāo)本中蛋白的降解問題出現(xiàn)在一向等點(diǎn)聚焦過程中,其原因可能是由于重泡脹液提供的堿性環(huán)境,使得某些酶類或小分子物質(zhì)得以激活;3)對(duì)十二指腸液1-DE圖譜進(jìn)行了分類與分析,共發(fā)現(xiàn)3種圖譜類型,其中的Ⅱ型圖譜可能是由于十二指腸液標(biāo)本中混入了胃液;4)建立了消化系統(tǒng)良性疾病患者的十二指腸液蛋白數(shù)據(jù)庫;5)十二指腸液可以起到輔助胰液蛋白質(zhì)組學(xué)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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