發(fā)布時間：2018-01-04 03:03

  本文關(guān)鍵詞：膀胱癌組織差異表達(dá)蛋白質(zhì)的篩選、鑒定及Derlin-1與膀胱癌臨床特征的相關(guān)性分析　出處：《南京醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 膀胱癌 質(zhì)譜分析 內(nèi)質(zhì)網(wǎng)相關(guān)降解蛋白1(Derlin-1) 上皮-間質(zhì)轉(zhuǎn)化(EMT)

【摘要】：目的:膀胱癌是泌尿外科臨床上最常見的惡性腫瘤之一。按照膀胱癌惡性程度不同,組織學(xué)分級可分為低級別癌和氋級別癌兩類;按照其浸潤深度可分為非肌層浸潤性和肌層浸潤性膀胱癌兩種。盡管臨床上各種手術(shù)方式及放化療手段不斷在提高和改進(jìn),但膀胱癌治療后的復(fù)發(fā)率仍然很高,五年生存率較低,特別是高級別肌層浸潤性癌的預(yù)后更差。探究膀胱癌的發(fā)病機理,尋找有價值的腫瘤標(biāo)記物和新的治療靶點,對于指導(dǎo)腫瘤的臨床治療,判斷腫瘤的預(yù)后具有重要的理論與現(xiàn)實意義。通過研究相關(guān)蛋白質(zhì)在腫瘤中的表達(dá)和功能,整體、動態(tài)、多角度的研究膀胱癌發(fā)生發(fā)展過程中的蛋白質(zhì)組的變化,篩選出與其發(fā)生發(fā)展相關(guān)的特征性蛋白質(zhì),可以尋找到具有特異性和敏感性的腫瘤標(biāo)記物,有助于膀胱癌的早期診斷、治療以及預(yù)后的判斷。方法:1.從江蘇省人民醫(yī)院泌尿外科收集144例膀胱癌組織標(biāo)本。選取3例男性患者的新鮮的高級別浸潤性膀胱移行上皮癌組織及癌旁組織,應(yīng)用Q Exactive質(zhì)譜儀和生物信息學(xué)方法進(jìn)行差異蛋白分析,初步篩選候選蛋白分子。2.隨機選取10對浸潤性膀胱癌和癌旁組織,應(yīng)用qRT-PCR、Western blot分別從mRNA和蛋白水平對候選分子Derlin-1在膀胱癌組織和癌旁組織中的表達(dá)差異進(jìn)行驗證。3.采用組織芯片結(jié)合免疫組化方法檢測Derlin-1蛋白在144例膀胱癌組織和31例癌旁組織中的表達(dá)情況;利用卡方檢驗分析Derlin-1陽性率與膀胱癌臨床特征的相關(guān)性。采用Kaplan-Meier法進(jìn)行生存分析,并用Log-rank檢驗組間差異。用COX比例風(fēng)險模型進(jìn)行多因素分析。4.體外細(xì)胞實驗,首先應(yīng)用Western Blot技術(shù)檢測Derlin-1蛋白在人膀胱上皮永生化細(xì)胞SV-HUC-1、膀胱癌細(xì)胞系UM-UC-3及T24中的表達(dá),然后應(yīng)用siRNA技術(shù)沉默Derlin-1在T24細(xì)胞中的表達(dá),應(yīng)用Transwell培養(yǎng)體系觀察轉(zhuǎn)染后的T24細(xì)胞遷移和侵襲能力的改變,同時應(yīng)用Western Blot和免疫熒光技術(shù)觀察EMT相關(guān)蛋白Vimentin、E-cadherin的表達(dá),探討Derlin-1在膀胱癌中可能的作用機制。結(jié)果:1.3例膀胱癌及癌旁組織經(jīng)質(zhì)譜分析,共篩選出165種差異蛋白,膀胱癌組織中上調(diào)表達(dá)19種、下調(diào)表達(dá)146種。經(jīng)生物信息學(xué)分析這些差異蛋白,其中DERL1(PREDICTED:derlin-1 isoform X1)對應(yīng) Derlin-1 蛋白,可能與膀胱癌發(fā)生有潛在關(guān)聯(lián)。還篩選出了一些其他的顯著差異表達(dá)基因ACTA2、ACTN1和VCL,以及焦點粘連通路和ECM受體相互作用等通路。從質(zhì)譜分析結(jié)果中選擇膀胱癌相關(guān)蛋白Derlin-1為候選蛋白分子。2.qRT-PCR檢測結(jié)果顯示,10例浸潤性膀胱癌組織中Derlin-1 mRNA水平是對應(yīng)的癌旁組織的2.4倍,明顯高于癌旁組織,差異有統(tǒng)計學(xué)意義(P0.05)。Western blot檢測結(jié)果顯示,浸潤性膀胱癌組織中Derlin-1蛋白相對表達(dá)量是癌旁組織的3.6倍,是非浸潤性膀胱癌組織的1.5倍,差異有統(tǒng)計學(xué)意義(P0.05)。3.組織芯片的免疫組化染色顯示,Derlin-1在浸潤性(98例)和非浸潤性(46例)膀骯癌中的陽性率分別為73.5%和47.8%,而癌旁組織(31例)陽性率為32.3%,三者差異有統(tǒng)計學(xué)意義(P0.001)。Derlin-1的陽性表達(dá)與患者年齡、性別無相關(guān)性(P0.05),與腫瘤分期、病理分級、淋巴結(jié)轉(zhuǎn)移均呈顯著相關(guān)性(P0.05)。Kaplan-Meier法分析顯示,Derlin-1陽性和陰性表達(dá)的膀胱癌患者中位生存期分別為46個月(95%CI:36.3-55.7月)和56個月(95%CI:51.4-60.6月);Derlin-1陽性表達(dá)的膀胱癌患者五年生存率明顯低于陰性患者,分別為21.79%和46.67%,兩者差異有統(tǒng)計學(xué)意義(P0.001)。COX多因素分析結(jié)果顯示,Derlin-1陽性、腫瘤分期以及淋巴結(jié)轉(zhuǎn)移是預(yù)測膀胱癌獨立預(yù)后因素(P0.05)。4.在體外細(xì)胞實驗中,Western Blot技術(shù)檢測Derlin-1蛋白在人膀胱上皮永生化細(xì)胞SV-HUC-1,膀胱癌細(xì)胞系UM-UC-3及T24中的表達(dá)結(jié)果顯示,Derlin-1蛋白在UM-UC-3、T24中相對表達(dá)量高于在SV-HUC-1中,尤其在T24細(xì)胞中表達(dá)更高,是UM-UC-3細(xì)胞中表達(dá)的6.646倍,是SV-HUC-1細(xì)胞中表達(dá)的20.038倍,三者間差異有統(tǒng)計學(xué)意義(P0.01)。T24細(xì)胞siRNA沉默Derlin-1基因表達(dá)后,Western Blot和免疫熒光結(jié)果顯示,Derlin-1蛋白表達(dá)量下降,與對照組的T24細(xì)胞相比,基因沉默后的T24細(xì)胞Vimentin蛋白低表達(dá),E-cadherin蛋白高表達(dá),差異有統(tǒng)計學(xué)意義(P0.001)。另外,Transwell培養(yǎng)體系結(jié)果顯示,基因沉默的T24細(xì)胞的遷移和侵襲數(shù)量低于未轉(zhuǎn)染的T24細(xì)胞,差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1.蛋白質(zhì)譜分析結(jié)合生物信息學(xué)分析方法在膀胱癌組織中篩選并鑒定出165種差異表達(dá)蛋白質(zhì),上調(diào)的蛋白有19種,下調(diào)的蛋白有146種。其中DERL1(PREDICTED:derlin-1 isoform X1)對應(yīng) Derlin-1 蛋白。2.Derlin-1在膀胱癌組織中高表達(dá),與膀胱癌的腫瘤分期、病理組織學(xué)分級以及淋巴結(jié)轉(zhuǎn)移有關(guān),陽性表達(dá)的患者五年生存率明顯降低。腫瘤分期、Derlin-1陽性以及淋巴結(jié)轉(zhuǎn)移是預(yù)測膀胱癌獨立預(yù)后因素。3.Derlin-1在膀胱癌細(xì)胞系中高表達(dá),Derlin-1在膀胱癌發(fā)生發(fā)展過程中的作用可能與EMT機制有關(guān)。Derlin-1有望成為潛在的膀胱癌診斷和預(yù)測預(yù)后的新的生物學(xué)靶點。
[Abstract]:Objective: bladder cancer is one of the most common malignant tumor of the clinical department of urology. According to the different malignant degree of bladder cancer, histological grade can be divided into low grade cancer and Meng grade cancer two; according to the depth of invasion can be divided into non muscle invasive and muscle invasive bladder cancer two. Although all kinds of surgery methods clinical and the chemotherapy means constantly improving and improving, but the treatment of bladder cancer recurrence rate is still high, the five year survival rate is low, especially the high level of muscle invasive cancer prognosis. The pathogenesis study of bladder cancer, tumor marker for valuable and new therapeutic targets for clinical treatment of tumors, it has important theoretical and practical significance in judging the prognosis of tumor. By studying the expression and function of proteins in tumors, the overall, dynamic, the development process of protein in bladder cancer research angle The change group, were correlated with the occurrence and development of characteristic proteins, can find tumor markers with specificity and sensitivity, it is helpful to the early diagnosis of bladder cancer, treatment and prognosis. Methods: 1. from the Department of Urology of Jiangsu Province People's Hospital collected 144 cases of bladder cancer tissue specimens from 3 cases of male patients. Fresh high grade invasive bladder transitional cell carcinoma tissues and adjacent tissues, differences in protein analysis method using Q Exactive mass spectrometry and bioinformatics, preliminary screening of candidate protein.2. randomly selected 10 of invasive bladder cancer and application of qRT-PCR, Western, mRNA and blot respectively from the protein level of the expression of candidate molecules of Derlin-1 in bladder cancer tissues and adjacent tissues of the difference.3. is validated by using tissue chip combined with immunohistochemical detection of Derlin-1 protein in 144 cases of bladder cancer Expression of tissue and 31 cases of adjacent tissues; the correlation analysis of clinical characteristics and the positive rate of Derlin-1 in bladder cancer by chi square test. Survival analysis was performed by Kaplan-Meier method, and Log-rank test the differences between groups..4. analysis of in vitro experiments using COX proportional hazards model for multivariate, the first application of the detection of Derlin-1 protein Western Blot in human bladder cell SV-HUC-1 expression and biochemical Pillon, bladder cancer cell lines UM-UC-3 and T24, and then the application of siRNA technology to silence Derlin-1 expression in T24 cells were observed in T24 system, cell migration and invasion ability changes after dyeing by Transwell culture, at the same time the application of EMT related protein Vimentin Western Blot and immunofluorescence observation technology, the expression of E-cadherin, explore the possible mechanisms of Derlin-1 in bladder carcinoma. Results: 1.3 cases of bladder carcinoma and paracancerous tissues were subjected to mass spectrometry analysis. Identified 165 different proteins in bladder cancer tissues 19 up-regulated, down regulated expression of 146. Bioinformatic analysis of these proteins, including DERL1 (PREDICTED:derlin-1 isoform X1) corresponding to Derlin-1 protein may have potential association with bladder cancer. The expression of ACTA2 gene also screened out some other significant differences, ACTN1 and VCL, as well as the focal adhesion pathway and ECM receptor interaction pathway. Selection of bladder cancer associated protein Derlin-1 from mass spectrometry analysis results show that as a candidate protein molecular testing results of.2.qRT-PCR and Derlin-1 mRNA levels in 10 cases of invasive bladder cancer is 2.4 times higher than paracancerous tissues, adjacent tissues, there were significant differences (P0.05).Western blot assay showed that the invasive bladder cancer tissues the expression of Derlin-1 protein is 3.6 times that of the adjacent tissue, non invasive bladder cancer 1.5 times group, the difference was statistically significant (P0.05) immunohistochemical.3. tissue microarray results in invasive Derlin-1 (98 cases) and non invasive (46 cases) positive rate of bladder cancer were 73.5% and 47.8%, and the adjacent tissues (31 cases) positive rate was 32.3%. There was a significant difference between the three groups (P0.001) positive.Derlin-1 expression and age, no correlation between gender (P0.05), and tumor staging, pathological grading, lymph node metastasis was significantly correlated (P0.05) analysis showed that the.Kaplan-Meier method, the expression of Derlin-1 positive and negative patients with bladder cancer. The median survival time was 46 months (95%CI:36.3-55.7 months) and 56 months (95%CI:51.4-60.6 months); the positive expression of Derlin-1 in bladder cancer patients five years survival rate was significantly lower than negative patients were 21.79% and 46.67%, the difference was statistically significant (P0.001) multivariate.COX analysis showed that Derlin-1 positive , tumor stage and lymph node metastasis is the independent prognostic factor for bladder cancer prediction (P0.05).4. in vitro, detect Derlin-1 protein Western Blot in human bladder on biochemical Pillon cell SV-HUC-1, the expression of bladder cancer cell lines UM-UC-3 and T24 showed that Derlin-1 protein in UM-UC-3, T24 relative expression quantity is higher than that in in SV-HUC-1, especially in T24 cells was 6.646 times higher, the expression of UM-UC-3 in cells, is 20.038 times the expression in SV-HUC-1 cells, there was statistically significant difference between the three (P0.01).T24 expression in siRNA cells after Derlin-1 gene silencing, Western Blot and immunofluorescence analysis showed that Derlin-1 protein expression decreased, compared with and the control group of T24 cells, the low expression of gene silencing in T24 cells after Vimentin protein, E-cadherin protein expression, the difference was statistically significant (P0.001). In addition, the results of Transwell training system Show that the number of migration and invasion of T24 cells in gene silencing than non transfected T24 cells, the difference was statistically significant (P0.05). Conclusion: 1. protein mass spectrometry combined with bioinformatics analysis method in bladder cancer screening and identified 165 differentially expressed proteins, 19 proteins up-regulated, down regulated protein 146. The DERL1 (PREDICTED:derlin-1 isoform X1) corresponding to Derlin-1 high expression of.2.Derlin-1 protein in bladder cancer, and bladder cancer staging, histological grade and lymph node metastasis, the positive expression of the five year survival rate of patients decreased significantly. Derlin-1 positive tumor staging, and lymph node metastasis are independent prognostic factors for predicting.3.Derlin-1 bladder cancer is highly expressed in bladder cancer cell line, Derlin-1 takes effect in the process of the development of EMT and related to the mechanism of.Derlin-1 in bladder cancer is expected to become Potential new biological targets for the diagnosis and prognosis of bladder cancer.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.14

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本文編號：1376742