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  本文關(guān)鍵詞：基于蛋白質(zhì)組學分析的良、惡性胸腔積液分子標志物搜尋　出處：《安徽醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 胸腔積液 蛋白質(zhì)組學 標志物 2-DE iTRAQ

【摘要】：目的良、惡性胸腔積液的鑒別診斷為臨床常見難題之一。本研究旨在通過蛋白質(zhì)組學分析技術(shù)搜尋良、惡性胸腔積液的分子標志物,并初步評估其用于二者鑒別診斷的臨床價值。方法研究先后采用了兩項蛋白質(zhì)組學技術(shù):一為雙向凝膠電泳(2-DE)技術(shù),二為相對和絕對定量同位素標記(iTRAQ)技術(shù)。第一條實驗路線采用雙向凝膠電泳在胸腔積液樣本中分離、搜尋蛋白,使用基質(zhì)輔助激光解吸飛行時間質(zhì)譜(MALDI-TOF-MS)鑒定差異表達蛋白的具體類型,并用ELISA法驗證候選蛋白標志物在良惡性胸腔積液樣本中的具體含量。第二條實驗路線采用基于iTRAQ的二維色譜串聯(lián)質(zhì)譜技術(shù)(iTRAQ-2D LC-MS/MS)在胸腔積液樣本中分離、搜尋蛋白并進行相對定量,對于差異蛋白基于基因本體(GO)數(shù)據(jù)庫進行GO富集分析(生物學過程、細胞組分及分子功能富集分析),通過KEGG信號通路分析(KEGG Pathway)分析尋找與鑒定差異蛋白關(guān)系最密切的病理通路,并構(gòu)建蛋白相互作用(PPI)網(wǎng)絡(luò)。在此基礎(chǔ)上進一步了解良、惡性胸腔積液蛋白組差異,并挑選有成為標志物潛力的蛋白。結(jié)果基于雙向凝膠電泳實驗路線,惡性組對比良性組,共發(fā)現(xiàn)明顯差異蛋白點(光密度值變化≥2倍)43個,上調(diào)9個,下調(diào)34個;對其中7個顯著差異點(光密度值變化≥3倍)質(zhì)譜鑒定明確了具體類型;挑選顯著差異點中免疫球蛋白λ(Igλ)、結(jié)合珠蛋白(Hp)進行ELISA驗證,結(jié)果表明Igλ在良、惡性胸腔積液中含量差異無統(tǒng)計學意義,Hp含量組間差異有統(tǒng)計學意義(P0.05)。進一步評估顯示診斷標準為胸腔積液中Hp≤389.02μg/L時,診斷惡性胸腔積液靈敏度為75.00%,特異度為52.38%。基于iTRAQ-2D LC-MS/MS實驗路線,共發(fā)現(xiàn)良、惡性胸腔積液差異蛋白151個(同位素標記相對定量變化≥1.2倍),其中上調(diào)84個,下調(diào)67個。GO分析顯示,二者在生物學過程上主要在單細胞、多細胞生物過程及應(yīng)激相關(guān)蛋白上存在差異;在分子功能上主要在蛋白結(jié)合功能上存在差異;在細胞組分上主要在胞外蛋白上存在差異。KEGG通路分析及蛋白互作網(wǎng)絡(luò)顯示,差異蛋白主要富集在補體與凝血級聯(lián)反應(yīng)通路,此外,還在磷脂酰肌醇3激酶/蛋白激酶B(PI3K-Akt)這條與腫瘤發(fā)生發(fā)展密切相關(guān)的信號通路富集。綜合考慮蛋白的差異變化程度以及蛋白間相互作用后,數(shù)據(jù)顯示α-烯醇化酶(ENO1)、磷酸甘油酸激酶1(PGK1)、生腱蛋白(TNC)最具有成為蛋白標志物的前景。結(jié)論基于雙向凝膠凝膠電泳技術(shù)搜尋到的標志物結(jié)合珠蛋白(Hp)有望用于良、惡性胸腔積液的輔助鑒別診斷;基于iTRAQ技術(shù),對于良、惡性胸腔積液在蛋白組成上的差異有了進一步了解,并搜尋到α-烯醇化酶(ENO1)、磷酸甘油酸激酶1(PGK1)、生腱蛋白(TNC)三個在良、惡性胸腔積液中明顯差異表達的蛋白�？傊�,蛋白組質(zhì)學技術(shù)的應(yīng)用,可望為良、惡性胸腔積液分子標志物的搜尋提供幫助,且iTRAQ技術(shù)較之雙向凝膠電泳技術(shù)可能更有優(yōu)勢。
[Abstract]:Objective the differential diagnosis of benign and malignant pleural effusion is one of the common clinical problems. The aim of this study is to search for molecular markers of benign and malignant pleural effusions by proteomic analysis, and to preliminarily evaluate the clinical value of these two markers in differential diagnosis. Two proteomics techniques have been adopted: one is two-dimensional gel electrophoresis (2-DE), the other is relative and absolute quantitative isotope labeling (iTRAQ). The first experimental line separation, search protein in pleural effusion samples by two-dimensional gel electrophoresis, using matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) specific types of protein expression were identified, and verify the candidate protein markers of specific content in benign and malignant pleural effusion samples by ELISA method. Second experimental route using tandem mass spectrometry technology based on two-dimensional chromatography iTRAQ (iTRAQ-2D LC-MS/MS) in isolation, pleural effusion samples for protein and relative quantitative differences in protein based on Gene Ontology (GO) GO enrichment analysis database (biological process, cellular component and molecular function enrichment analysis), through KEGG pathway analysis (KEGG Pathway) for protein analysis and identification of differences are most closely related to the pathological pathway, and construct protein interaction network (PPI). On this basis, the differences in protein groups of benign and malignant pleural effusion were further studied, and the protein that had potential as a marker was selected. Based on the results of the experimental line of two-dimensional gel electrophoresis, malignant group and benign group comparison, were found significantly different protein spots (optical density change is more than 2 times) 43, up 9, down 34; 7 of them significantly differences (optical density change is more than 3 times) mass spectrometry to identify clearly the specific types of immune; globulin significant differences in selection of lambda (Ig x), haptoglobin (Hp) ELISA verification, the results show no significant difference in the content of benign and malignant pleural effusion Ig, there were statistically significant differences between the groups in Hp (P0.05). Further evaluation showed that the criteria for the diagnosis of pleural effusion in Hp is less than or equal to 389.02 g/L, the sensitivity of diagnosis of malignant pleural effusion was 75%, the specificity was 52.38%. ITRAQ-2D LC-MS/MS based on the experimental route, were found in benign and malignant pleural effusion (151 differential protein isotope labeling relative quantitative change is more than 1.2 times), which increased by 84, down 67. GO analysis showed that there were differences in biological processes between single cells, multicellular biological processes and stress related proteins in two organisms. There were differences in molecular function between protein and binding function. KEGG pathway analysis and protein interaction network showed that the differential proteins were mainly concentrated in complement and coagulation cascade pathway. In addition, phosphatidylinositol 3 kinase / protein kinase B (PI3K-Akt) was also enriched in signal pathways closely related to tumor development. Considering the interaction of protein and protein changes in the degree of difference between after data showed that alpha enolase (ENO1) and phosphoglycerate kinase 1 (PGK1), tenascin (TNC) has become the most protein markers in prospect. Conclusion two dimensional gel electrophoresis markers to search based on haptoglobin (Hp) is expected to be used in differential diagnosis of benign and malignant pleural effusion; based on the technology of iTRAQ for benign and malignant pleural effusion with the further understanding of differences in protein composition, and to search for alpha enolase (ENO1), phosphoglycerate kinase 1 (PGK1), tenascin (TNC) three protein expression difference in benign and malignant pleural effusion. In conclusion, the application of proteomic technology is expected to provide help for searching for molecular markers in benign and malignant pleural effusion, and iTRAQ technology is more advantageous than two-dimensional gel electrophoresis.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R561.3

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本文編號：1338228