触珠蛋白和补体因子H在胆道肿瘤中的诊断价值

发布时间：2018-05-08 23:01

本文选题：胆道肿瘤 + 触珠蛋白　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究目的胆道肿瘤是来源于胆道和胆囊上皮的胆管细胞分化而来的一组异型性肿瘤,可分为胆管癌,胆囊癌和壶腹部肿瘤,是致死率最高的肿瘤之一。肿瘤根治性切除术往往能取得较理想的结果,然而,这些肿瘤常诊断较晚,可手术率低。CA19-9是目前诊断胆道肿瘤最常用的生物标志物,也是迄今为止最有前景的胆道肿瘤的诊断及预后标志。然而,在胆汁淤积及胆管炎患者中,CA19-9水平亦升高,降低了恶性肿瘤诊断的特异性。因此,探寻胆道肿瘤早期诊断的生物标志物是目前临床上亟需解决的问题。血液是目前最常用的检测对象,然而,其中富含的10种蛋白含量大于其总蛋白的90%,这些富含的蛋白质包含了很少的代表器官状态的信息,而且极大地抑制了可能包含更多信息的低丰度蛋白的精确检测。肿瘤细胞可能直接释放和/或脱落蛋白至胆汁,使得胆汁中分泌或脱落的生物标志物水平高于血清,并且胆汁中富含糖蛋白。因而,本研究将胆汁作为主要的分析对象。本研究经过对比恶性胆道肿瘤与良性胆道疾病患者胆汁的蛋白组表达差别,探究胆道肿瘤潜在的生物标志物,为胆道肿瘤的早期诊断提供新方向,同时为进一步的肿瘤治疗提供可能的靶点。研究方法蛋白质组学是对生物样品中蛋白质和多肽的识别和定量分析,是研究蛋白质的表达和活性的一门学科。蛋白质组学能全面、动态、定量地观察肿瘤进展和转移过程中蛋白表达的变化。同重同位素相对与绝对定量(Isobaric tags for relative and absolute quantitation,i TRAQ)标记联合二维液相色谱串联质谱分析(2D-LC-MS/MS)是新进的探寻肿瘤标志物蛋白质组学方法的强有力技术措施之一。iTRAQ技术是一种在定量蛋白质组学领域最重要的技术,可用于多达八个样品在一个实验中的定量分析。Kyoto Encyclopedia of Genes and Genomes(KEGG)数据库是有关Pathway的主要公共数据库,经过Pathway分析能确定蛋白质介入的最主要生化代谢途径和信号转导路线。Gene Ontology(GO)是一个国际标准化的基因功能分类体制,提供了一套动态革新的标准词汇表来全面描述生物体中基因和基因产物的属性。GO总共有三个本体,分别描述基因的分子功能(Molecular Function,MF)、所处的细胞位置(Cellular Component,CC)、参与的生物过程(Biological Process,BP)。收集经病理结果证实的恶性胆道肿瘤和良性胆道疾病患者的胆汁、血清标本,离心后-80℃保存,并记录病人的相关临床资料。胆汁蛋白是从随机抽取的8例患者胆汁中提取的总蛋白,消化后的肽段用稳定同位素试剂标记,将肽混合物行离线2D-LC分离,然后进行Triplet TOF 5600检测,Mascot和scaffold进行定性定量,最后进行生物信息学分析(KEGG通路分析、GO分析、PPI蛋白相互作用网络分析)。同时,选取胆道肿瘤可能的候选生物标志物,分别对50例恶性胆道肿瘤患者和50例良性胆道疾病患者进行胆汁和血清的Western blot、ELISA验证,并通过ROC曲线确定高表达蛋白诊断的敏感性和特异性,从而明确其在胆道肿瘤中的诊断价值。运用Kaplan-Meier生存曲线和Cox分析,评价胆道肿瘤和胆管结石患者中可能生物标志物的预后价值。结果4组蛋白标本2D LC-MS/MS质谱检测出10个差异表达的蛋白(FDR1%),6个蛋白表达下调,4个蛋白表达上调,其中触珠蛋白(Hp)和补体因子H(CFH)在胆道肿瘤组的表达明显上调。Western blot分析结果表明,触珠蛋白在恶性肿瘤组胆汁和血清中高表达。ELISA结果提示,胆汁中Hp、CFH蛋白浓度明显高于血清(P=0.004);触珠蛋白和补体因子H在恶性肿瘤组胆汁中表达升高(P值分别为0.006和0.014),不同类型胆道肿瘤中表达无明显差异;而血清样本中,胆管癌和胆囊癌触珠蛋白、补体因子H浓度明显升高,而十二指肠乳头癌则明显降低(P值分别为0.033和0.004)。ROC曲线分析发现,当Hp=57.237 ng/ml时,其敏感性为0.909,特异性为0.667;当CFH=148.1807 ng/ml时,其敏感性为0.636,特异性为0.8。根据ROC曲线所得的临界值,将恶性胆道肿瘤组患者血清触珠蛋白及补体因子H分为阳性和阴性两大类。Kaplan-Meier曲线提示患者预后与触珠蛋白和补体因子H表达密切相关(P值分别为0.001和0.002),触珠蛋白、补体因子H表达阳性较阴性患者预后差;患者较长的生存期与蛋白表达双阴性有关(P值分别为0.003、0.031及0.02),任意两种肿瘤标志物表达均为阴性患者预后最好,均为阳性表达患者预后最差,有且仅有一种表达为阳性者预后居两者之间。结论触珠蛋白和补体因子H在胆道恶性肿瘤中表达上调,并且与患者预后密切相关,其中触珠蛋白诊断敏感性高于CA19-9和CEA,大概为胆道肿瘤潜在的生物标志物及治疗靶点。上调的触珠蛋白是否存在着糖基化,不同细胞来源的胆道肿瘤是否存在着糖连接方式以及结合位点的差异还需进一步研究。
[Abstract]:Objective bile duct tumor is a group of heteromorphic tumors derived from bile duct and gallbladder epithelial cells, which can be divided into bile duct cancer, gallbladder cancer and ampullary tumor. It is one of the most fatal tumors. Radical resection of tumors often results in more ideal results. However, these tumors are often diagnosed late and have low operative rate. CA19-9 is the most commonly used biomarker for the diagnosis of biliary tumors, and it is also the most promising diagnostic and prognostic marker of biliary tract tumors to date. However, in patients with cholestasis and cholangitis, the level of CA19-9 is also elevated, reducing the specificity of the diagnosis of malignant tumors. Therefore, the biomarkers for the early diagnosis of biliary tumors are the objectives. Blood is the most commonly used problem in the clinic. Blood is the most commonly used detection object at present. However, the content of 10 proteins is more than 90% of its total protein. These rich proteins contain little information about the state of the organ, and it greatly inhibits the accurate detection of low abundance proteins that may contain more information. The cells may directly release and / or exfoliate to bile, making the level of biomarkers in the bile secreted or exfoliated higher than in the serum, and the bile is rich in glycoproteins. Therefore, this study uses the bile as the main object of analysis. Exploring the potential biomarkers of biliary tumors, providing new directions for the early diagnosis of biliary tumors and providing possible targets for further cancer treatment. Proteomics is a study of the identification and quantitative analysis of proteins and peptides in biological samples. It is a subject for the study of the expression and activity of egg white matter. The study can comprehensively, dynamically and quantitatively observe the changes in protein expression during tumor progression and metastasis. Tandem mass spectrometry (Isobaric tags for relative and absolute quantitation, I TRAQ) labeling combined with two dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/ MS) is a new proteomic approach for exploring tumor markers. One of the powerful technical measures,.ITRAQ technology, is one of the most important techniques in the field of quantitative proteomics, which can be used in the quantitative analysis of up to eight samples in one experiment. The.Kyoto Encyclopedia of Genes and Genomes (KEGG) database is the main public data base about Pathway, and the protein intervention can be determined through Pathway analysis. .Gene Ontology (GO), the main biochemical metabolic pathway and signal transduction pathway, is an internationally standardized gene functional classification system. It provides a dynamic and innovative standard vocabulary to describe the properties of gene and gene products in a total of three bodies, describing the molecular function of the gene (Molecular Functio, respectively). N, MF), the location of the cell (Cellular Component, CC), the bioprocess (Biological Process, BP) involved in the collection of bile, serum specimens of patients with malignant biliary and benign biliary diseases confirmed by pathological results, preserved at -80 at -80, and recorded the related clinical data of the sick people. Bile protein is the 8 patients' bile extracted from random. The total protein extracted from the juice, the peptide segment after digestion was marked with stable isotope reagent, the peptide mixture was separated by off-line 2D-LC, then Triplet TOF 5600 was detected, Mascot and scaffold were qualitatively and quantified. Finally, bioinformatics analysis (KEGG pathway analysis, GO analysis, PPI protein interaction network analysis). At the same time, the bile duct swelling was selected. The possible candidate biomarkers of the tumor were tested for 50 cases of malignant biliary tumors and 50 cases of benign biliary disease by Western blot, ELISA, and the sensitivity and specificity of the diagnosis of high expression proteins by ROC curve, so that the diagnostic value of the high expression protein in the biliary tract tumor was determined. The Kaplan-Meier survival curve was used. The prognostic value of possible biomarkers in patients with biliary and bile duct stones was evaluated by line and Cox analysis. Results 10 differentially expressed proteins (FDR1%) were detected in 4 groups of protein specimens by 2D LC-MS/MS mass spectrometry, 6 protein expressions were downregulated and 4 proteins were up-regulated, in which the expression of Hp and complement factor H (CFH) in the biliary tract tumor group was obviously expressed. The up-regulated.Western blot analysis showed that the high expression of.ELISA in the bile and serum of the malignant tumor group showed that the concentration of Hp and CFH protein in the bile was significantly higher than that in the serum (P=0.004), and the expression of TDC and complement factor H increased in the bile of the malignant tumor group (P value was 0.006 and 0.014 respectively), and expressed in different types of biliary tumors. There was no significant difference in the serum samples of cholangiocarcinoma and gallbladder cancer, the concentration of complement factor H increased significantly, while the duodenal papilla carcinoma decreased significantly (P value 0.033 and 0.004).ROC curve analysis found that when Hp=57.237 ng/ml, the sensitivity was 0.909, specificity 0.667, and when CFH=148.1807 ng/ml, the sensitivity was 0.636, The specificity was 0.8. based on the critical value of the ROC curve, and the serum tactile protein and complement factor H in the malignant biliary tumor group were divided into two kinds of.Kaplan-Meier curves, positive and negative, suggesting that the prognosis was closely related to the expression of TDC and complement factor H (P value was 0.001 and 0.002 respectively), and tactile protein and complement factor H expression were positive. The prognosis of the more negative patients was poor; the longer survival time of the patients was related to the double negative protein expression (P value was 0.003,0.031 and 0.02), and the expression of any two tumor markers was the best in the negative patients, and the prognosis was the worst in the positive expression patients, and the prognosis of the only one expression was between the two. The expression of factor H is up-regulated in the malignant tumor of the biliary tract, and is closely related to the prognosis of the patients. The diagnostic sensitivity of the TD is higher than that of CA19-9 and CEA. It is probably a potential biomarker and target for the treatment of biliary tract tumors. The difference between the binding site and the binding site should be further studied.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.8

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