【摘要】：目的:本研究通過蛋白質(zhì)組學(xué)技術(shù)篩選正常膀胱組織與結(jié)核性攣縮膀胱組織、正常輸尿管組織與結(jié)核性狹窄輸尿管組織標(biāo)本之間共有的差異蛋白,查閱文獻(xiàn)并從差異蛋白中篩選可能與膀胱及輸尿管纖維化病變發(fā)生、發(fā)展的相關(guān)蛋白,通過蛋白質(zhì)免疫印跡技術(shù)進(jìn)行差異蛋白驗(yàn)證,為膀胱攣縮及輸尿管狹窄的分級(jí)、診斷、治療提供新的方法及靶點(diǎn)。方法:第一部分:收集2015年1月至2016年12月解放軍309醫(yī)院泌尿外科確診泌尿系結(jié)核患者的攣縮膀胱及狹窄輸尿管組織各2塊;獲取我院同一時(shí)期DCD供者的膀胱及輸尿管組織各2塊作為正常對(duì)照。4種組織混合后用iTRAQ標(biāo)記技術(shù)(相對(duì)和絕對(duì)定量同位素標(biāo)記)聯(lián)合LC-MSMS(液相色譜串聯(lián)質(zhì)譜)對(duì)各組的總蛋白進(jìn)分析,將在正常膀胱與攣縮膀胱、正常輸尿管與狹窄輸尿管組織中表達(dá)差異在2倍及2倍以上的蛋白質(zhì)定義為差異蛋白,并篩選2組之間共同表達(dá)的差異蛋白。通過Uniprot、DAVID、String、Wego數(shù)據(jù)庫對(duì)共有差異蛋白進(jìn)行功能聚類、細(xì)胞定位、相互作用分析。第二部分:分別選取上述4種組織各6塊,進(jìn)行Masson染色,驗(yàn)證攣縮膀胱、狹窄輸尿管組織膠原纖維的表達(dá)量;從共有的差異蛋白中挑選微纖絲相關(guān)糖蛋白4(MFAP4)和纖維調(diào)節(jié)素(FMOD)進(jìn)行蛋白質(zhì)免疫印跡(Western blot)驗(yàn)證。結(jié)果:第一部分:通過蛋白質(zhì)組學(xué)相關(guān)實(shí)驗(yàn),在正常膀胱組織與攣縮膀胱組織組共鑒定出188種差異蛋白質(zhì),正常輸尿管與狹窄輸尿管組共鑒定出130種差異蛋白質(zhì),2組所共有的差異蛋白質(zhì)48種,其中20種差異蛋白質(zhì)下調(diào),28種蛋白質(zhì)表達(dá)上調(diào);48種差異蛋白質(zhì)在DAVID數(shù)據(jù)庫中共有28種蛋白質(zhì)被標(biāo)記,這28種蛋白質(zhì)主要涉及17種生物學(xué)過程,5種分子功能途徑和13種細(xì)胞組成;48種蛋白質(zhì)在String數(shù)據(jù)庫中,39種蛋白質(zhì)被檢測到,共形成3條相互作用網(wǎng)絡(luò)。第二部分:Masson染色提示在攣縮膀胱組織與狹窄輸尿管組織中,肌層可見大量膠原纖維表達(dá),攣縮膀胱與正常膀胱、狹窄輸尿管與正常輸尿管相比,膠原纖維表達(dá)明顯增高,差異具有統(tǒng)計(jì)學(xué)意義;western blot結(jié)果提示MFAP4、FMOD在攣縮膀胱及狹窄輸尿管中表達(dá)明顯增高,差異具有統(tǒng)計(jì)學(xué)意義。結(jié)論:1、iTRAQ技術(shù)聯(lián)合LC-MSMS可對(duì)樣本蛋白進(jìn)行定性、定量鑒定,篩選出差異蛋白,并對(duì)蛋白進(jìn)行生物信息學(xué)分析;2、纖維調(diào)節(jié)素蛋白(FMOD)在病變組織中表達(dá)上調(diào),是影響膠原纖維合成的重要因素,與膀胱及輸尿管纖維化關(guān)系密切。3、人微纖絲相關(guān)糖蛋白4(MFAP4)在病變組織中高表達(dá),可進(jìn)一步行實(shí)驗(yàn)探討其在結(jié)核性纖維化病變的診斷、分級(jí)及預(yù)后中的運(yùn)用。
[Abstract]:Objective: to screen the common differential proteins between normal bladder tissue and tuberculous contracture bladder tissue, normal ureteral tissue and tuberculous stricture ureter tissue by proteomics. Literature was reviewed and proteins related to the occurrence and development of bladder and ureteral fibrosis were screened from the differential proteins. The differential protein was verified by Western blotting, which was the classification and diagnosis of bladder contracture and ureteral stricture. Treatment provides new methods and targets. Methods: the first part: from January 2015 to December 2016, 2 pieces of contracture bladder and 2 pieces of ureteral stricture were collected from urological department of PLA 309 Hospital. The bladder and ureter tissues of DCD donors in the same period were obtained as normal control. The four tissues were mixed with iTRAQ labeling technique (relative and absolute quantitative isotope labeling) combined with LC-MSMS (liquid chromatographic tandem substance). Analysis of the total protein in each group, The differential protein was defined as differential protein in normal bladder and contracture bladder, normal ureter and narrow ureteral tissue, and the differentially expressed proteins were screened between the two groups. Functional clustering, cellular localization and interaction analysis of common differentially expressed proteins were carried out by Uniprot,DAVID,String,Wego database. The second part: 6 pieces of each of the four tissues were selected for Masson staining to verify the expression of collagen fibers in contracture bladder and ureter. Microfibril associated glycoprotein 4 (MFAP4) and fibronectin (FMOD) were selected from common differential proteins for Western blot (Western blot) verification. Results: in the first part, 188 different proteins were identified in normal bladder tissue and contracture bladder tissue group, 130 differential proteins were identified in normal ureter and narrow ureter group. There were 48 differentially expressed proteins in both groups, of which 20 were down-regulated and 28 were up-regulated. A total of 28 proteins were labeled in the DAVID database. The 28 proteins were mainly involved in 17 biological processes, 5 molecular functional pathways and 13 cell compositions. In String database, 39 proteins were detected and 3 interaction networks were formed. In the second part, Masson staining showed that a large number of collagen fibers were expressed in the muscle layer of contracture bladder and narrow ureter, and the expression of collagen fibers in contracture bladder and normal bladder, narrow ureter and normal ureter were higher than those in normal ureter. The difference is statistically significant. Western blot results suggested that the expression of MFAP4,FMOD in contracture bladder and ureter was significantly higher than that in ureter. Conclusion: 1ITRAQ combined with LC-MSMS can identify the sample protein qualitatively and quantitatively, screen out the differential protein, and analyze the protein by bioinformatics. 2, the expression of fibronectin (FMOD) is up-regulated in the pathological tissues, which is an important factor affecting the synthesis of collagen fibers, and is closely related to the fibrosis of bladder and ureter. Human microfibril associated glycoprotein 4 (MFAP4) is highly expressed in pathological tissues, which can be used in the diagnosis, grading and prognosis of tuberculous fibrosis.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R699

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