基于蛋白质组学分析的良、恶性胸腔积液分子标志物搜寻

发布时间：2017-12-26 17:40

本文关键词：基于蛋白质组学分析的良、恶性胸腔积液分子标志物搜寻　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的良、恶性胸腔积液的鉴别诊断为临床常见难题之一。本研究旨在通过蛋白质组学分析技术搜寻良、恶性胸腔积液的分子标志物,并初步评估其用于二者鉴别诊断的临床价值。方法研究先后采用了两项蛋白质组学技术:一为双向凝胶电泳(2-DE)技术,二为相对和绝对定量同位素标记(iTRAQ)技术。第一条实验路线采用双向凝胶电泳在胸腔积液样本中分离、搜寻蛋白,使用基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)鉴定差异表达蛋白的具体类型,并用ELISA法验证候选蛋白标志物在良恶性胸腔积液样本中的具体含量。第二条实验路线采用基于iTRAQ的二维色谱串联质谱技术(iTRAQ-2D LC-MS/MS)在胸腔积液样本中分离、搜寻蛋白并进行相对定量,对于差异蛋白基于基因本体(GO)数据库进行GO富集分析(生物学过程、细胞组分及分子功能富集分析),通过KEGG信号通路分析(KEGG Pathway)分析寻找与鉴定差异蛋白关系最密切的病理通路,并构建蛋白相互作用(PPI)网络。在此基础上进一步了解良、恶性胸腔积液蛋白组差异,并挑选有成为标志物潜力的蛋白。结果基于双向凝胶电泳实验路线,恶性组对比良性组,共发现明显差异蛋白点(光密度值变化≥2倍)43个,上调9个,下调34个;对其中7个显著差异点(光密度值变化≥3倍)质谱鉴定明确了具体类型;挑选显著差异点中免疫球蛋白λ(Igλ)、结合珠蛋白(Hp)进行ELISA验证,结果表明Igλ在良、恶性胸腔积液中含量差异无统计学意义,Hp含量组间差异有统计学意义(P0.05)。进一步评估显示诊断标准为胸腔积液中Hp≤389.02μg/L时,诊断恶性胸腔积液灵敏度为75.00%,特异度为52.38%。基于iTRAQ-2D LC-MS/MS实验路线,共发现良、恶性胸腔积液差异蛋白151个(同位素标记相对定量变化≥1.2倍),其中上调84个,下调67个。GO分析显示,二者在生物学过程上主要在单细胞、多细胞生物过程及应激相关蛋白上存在差异;在分子功能上主要在蛋白结合功能上存在差异;在细胞组分上主要在胞外蛋白上存在差异。KEGG通路分析及蛋白互作网络显示,差异蛋白主要富集在补体与凝血级联反应通路,此外,还在磷脂酰肌醇3激酶/蛋白激酶B(PI3K-Akt)这条与肿瘤发生发展密切相关的信号通路富集。综合考虑蛋白的差异变化程度以及蛋白间相互作用后,数据显示α-烯醇化酶(ENO1)、磷酸甘油酸激酶1(PGK1)、生腱蛋白(TNC)最具有成为蛋白标志物的前景。结论基于双向凝胶凝胶电泳技术搜寻到的标志物结合珠蛋白(Hp)有望用于良、恶性胸腔积液的辅助鉴别诊断;基于iTRAQ技术,对于良、恶性胸腔积液在蛋白组成上的差异有了进一步了解,并搜寻到α-烯醇化酶(ENO1)、磷酸甘油酸激酶1(PGK1)、生腱蛋白(TNC)三个在良、恶性胸腔积液中明显差异表达的蛋白。总之,蛋白组质学技术的应用,可望为良、恶性胸腔积液分子标志物的搜寻提供帮助,且iTRAQ技术较之双向凝胶电泳技术可能更有优势。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R561.3

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