本文選題：膽道腫瘤 + 觸珠蛋白��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究目的膽道腫瘤是來(lái)源于膽道和膽囊上皮的膽管細(xì)胞分化而來(lái)的一組異型性腫瘤,可分為膽管癌,膽囊癌和壺腹部腫瘤,是致死率最高的腫瘤之一。腫瘤根治性切除術(shù)往往能取得較理想的結(jié)果,然而,這些腫瘤常診斷較晚,可手術(shù)率低。CA19-9是目前診斷膽道腫瘤最常用的生物標(biāo)志物,也是迄今為止最有前景的膽道腫瘤的診斷及預(yù)后標(biāo)志。然而,在膽汁淤積及膽管炎患者中,CA19-9水平亦升高,降低了惡性腫瘤診斷的特異性。因此,探尋膽道腫瘤早期診斷的生物標(biāo)志物是目前臨床上亟需解決的問(wèn)題。血液是目前最常用的檢測(cè)對(duì)象,然而,其中富含的10種蛋白含量大于其總蛋白的90%,這些富含的蛋白質(zhì)包含了很少的代表器官狀態(tài)的信息,而且極大地抑制了可能包含更多信息的低豐度蛋白的精確檢測(cè)。腫瘤細(xì)胞可能直接釋放和/或脫落蛋白至膽汁,使得膽汁中分泌或脫落的生物標(biāo)志物水平高于血清,并且膽汁中富含糖蛋白。因而,本研究將膽汁作為主要的分析對(duì)象。本研究經(jīng)過(guò)對(duì)比惡性膽道腫瘤與良性膽道疾病患者膽汁的蛋白組表達(dá)差別,探究膽道腫瘤潛在的生物標(biāo)志物,為膽道腫瘤的早期診斷提供新方向,同時(shí)為進(jìn)一步的腫瘤治療提供可能的靶點(diǎn)。研究方法蛋白質(zhì)組學(xué)是對(duì)生物樣品中蛋白質(zhì)和多肽的識(shí)別和定量分析,是研究蛋白質(zhì)的表達(dá)和活性的一門(mén)學(xué)科。蛋白質(zhì)組學(xué)能全面、動(dòng)態(tài)、定量地觀察腫瘤進(jìn)展和轉(zhuǎn)移過(guò)程中蛋白表達(dá)的變化。同重同位素相對(duì)與絕對(duì)定量(Isobaric tags for relative and absolute quantitation,i TRAQ)標(biāo)記聯(lián)合二維液相色譜串聯(lián)質(zhì)譜分析(2D-LC-MS/MS)是新進(jìn)的探尋腫瘤標(biāo)志物蛋白質(zhì)組學(xué)方法的強(qiáng)有力技術(shù)措施之一。iTRAQ技術(shù)是一種在定量蛋白質(zhì)組學(xué)領(lǐng)域最重要的技術(shù),可用于多達(dá)八個(gè)樣品在一個(gè)實(shí)驗(yàn)中的定量分析。Kyoto Encyclopedia of Genes and Genomes(KEGG)數(shù)據(jù)庫(kù)是有關(guān)Pathway的主要公共數(shù)據(jù)庫(kù),經(jīng)過(guò)Pathway分析能確定蛋白質(zhì)介入的最主要生化代謝途徑和信號(hào)轉(zhuǎn)導(dǎo)路線。Gene Ontology(GO)是一個(gè)國(guó)際標(biāo)準(zhǔn)化的基因功能分類(lèi)體制,提供了一套動(dòng)態(tài)革新的標(biāo)準(zhǔn)詞匯表來(lái)全面描述生物體中基因和基因產(chǎn)物的屬性。GO總共有三個(gè)本體,分別描述基因的分子功能(Molecular Function,MF)、所處的細(xì)胞位置(Cellular Component,CC)、參與的生物過(guò)程(Biological Process,BP)。收集經(jīng)病理結(jié)果證實(shí)的惡性膽道腫瘤和良性膽道疾病患者的膽汁、血清標(biāo)本,離心后-80℃保存,并記錄病人的相關(guān)臨床資料。膽汁蛋白是從隨機(jī)抽取的8例患者膽汁中提取的總蛋白,消化后的肽段用穩(wěn)定同位素試劑標(biāo)記,將肽混合物行離線2D-LC分離,然后進(jìn)行Triplet TOF 5600檢測(cè),Mascot和scaffold進(jìn)行定性定量,最后進(jìn)行生物信息學(xué)分析(KEGG通路分析、GO分析、PPI蛋白相互作用網(wǎng)絡(luò)分析)。同時(shí),選取膽道腫瘤可能的候選生物標(biāo)志物,分別對(duì)50例惡性膽道腫瘤患者和50例良性膽道疾病患者進(jìn)行膽汁和血清的Western blot、ELISA驗(yàn)證,并通過(guò)ROC曲線確定高表達(dá)蛋白診斷的敏感性和特異性,從而明確其在膽道腫瘤中的診斷價(jià)值。運(yùn)用Kaplan-Meier生存曲線和Cox分析,評(píng)價(jià)膽道腫瘤和膽管結(jié)石患者中可能生物標(biāo)志物的預(yù)后價(jià)值。結(jié)果4組蛋白標(biāo)本2D LC-MS/MS質(zhì)譜檢測(cè)出10個(gè)差異表達(dá)的蛋白(FDR1%),6個(gè)蛋白表達(dá)下調(diào),4個(gè)蛋白表達(dá)上調(diào),其中觸珠蛋白(Hp)和補(bǔ)體因子H(CFH)在膽道腫瘤組的表達(dá)明顯上調(diào)。Western blot分析結(jié)果表明,觸珠蛋白在惡性腫瘤組膽汁和血清中高表達(dá)。ELISA結(jié)果提示,膽汁中Hp、CFH蛋白濃度明顯高于血清(P=0.004);觸珠蛋白和補(bǔ)體因子H在惡性腫瘤組膽汁中表達(dá)升高(P值分別為0.006和0.014),不同類(lèi)型膽道腫瘤中表達(dá)無(wú)明顯差異;而血清樣本中,膽管癌和膽囊癌觸珠蛋白、補(bǔ)體因子H濃度明顯升高,而十二指腸乳頭癌則明顯降低(P值分別為0.033和0.004)。ROC曲線分析發(fā)現(xiàn),當(dāng)Hp=57.237 ng/ml時(shí),其敏感性為0.909,特異性為0.667;當(dāng)CFH=148.1807 ng/ml時(shí),其敏感性為0.636,特異性為0.8。根據(jù)ROC曲線所得的臨界值,將惡性膽道腫瘤組患者血清觸珠蛋白及補(bǔ)體因子H分為陽(yáng)性和陰性兩大類(lèi)。Kaplan-Meier曲線提示患者預(yù)后與觸珠蛋白和補(bǔ)體因子H表達(dá)密切相關(guān)(P值分別為0.001和0.002),觸珠蛋白、補(bǔ)體因子H表達(dá)陽(yáng)性較陰性患者預(yù)后差;患者較長(zhǎng)的生存期與蛋白表達(dá)雙陰性有關(guān)(P值分別為0.003、0.031及0.02),任意兩種腫瘤標(biāo)志物表達(dá)均為陰性患者預(yù)后最好,均為陽(yáng)性表達(dá)患者預(yù)后最差,有且僅有一種表達(dá)為陽(yáng)性者預(yù)后居兩者之間。結(jié)論觸珠蛋白和補(bǔ)體因子H在膽道惡性腫瘤中表達(dá)上調(diào),并且與患者預(yù)后密切相關(guān),其中觸珠蛋白診斷敏感性高于CA19-9和CEA,大概為膽道腫瘤潛在的生物標(biāo)志物及治療靶點(diǎn)。上調(diào)的觸珠蛋白是否存在著糖基化,不同細(xì)胞來(lái)源的膽道腫瘤是否存在著糖連接方式以及結(jié)合位點(diǎn)的差異還需進(jìn)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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.8

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