本文選題：腎小球 + 腎髓質��； 參考：《北京協(xié)和醫(yī)學院》2017年博士論文

【摘要】：研究背景:基于質譜的蛋白質組學技術已成為探索腎臟疾病發(fā)病機制及某些疑難腎臟疾病診斷的方法之一。目前利用激光顯微切割聯(lián)合質譜(Laser microdissection and mass spectromy,LMD/MS)技術對人腎小球進行蛋白質表達譜的研究有限,對腎髓質進行蛋白質組學表達譜的研究尚未見發(fā)表。采用LMD/MS技術不僅可分別獲取腎小球及腎髓質組織以構建蛋白表達譜,也可對腎病變組織區(qū)域進行分析。近年來,利用LMD/MS技術發(fā)現(xiàn)一些新的淀粉樣變亞型,使得淀粉樣變分型診斷水平有所提高;同時對致淀粉樣變蛋白進行氨基酸序列分析有可能發(fā)現(xiàn)變異的致淀粉樣變蛋白。研究方法:在第一部分工作中,我們收集6例癌旁腎組織,通過LMD/MS技術獲取腎小球及腎髓質組織并構建蛋白質組表達譜;對兩個部位所鑒定到的蛋白進行生物信息學分析。在第二部分工作中,我們分析了近5年45例AL型淀粉樣變性病患者臨床、實驗室檢查及腎活檢病理結果,選取20例腎組織免疫熒光分型不明確的患者進行免疫組化及LMD/MS分型,評價免疫組化及LMD/MS在免疫熒光分型不明確的AL淀粉樣變患者中的診斷意義。在第三部分工作中,對9例臨床、實驗室檢查及腎組織免疫化學檢查均不能分型的疑難腎淀粉樣變患者進行LMD/MS分析,以期達到腎淀粉樣變的完整分型診斷,并利用生物信息學方法對AA型淀粉樣變病例的SAA蛋白進行變異氨基酸序列分析。研究結果:第一部分中,腎小球及腎髓質分別鑒定到1373及1673種蛋白;與以往研究比較,本研究單獨鑒定到472種腎小球蛋白,并首次建立了正常人類腎髓質蛋白質組表達譜。生物信息學分析顯示腎小球蛋白多為細胞骨架蛋白,腎髓質蛋白多參與細胞代謝、具有酶解功能。第二部分中,我們發(fā)現(xiàn)應用免疫組化方法可對免疫熒光不能分型的部分AL型淀粉樣變進行分型診斷,敏感性約為55%;對于部分免疫組化方法不能分型的AL型淀粉樣變患者,LMD/MS技術仍可進行分型診斷;20例行LMD/MS分析的患者中,18例可明確分型診斷,敏感性為90%。第三部分中,9例疑難腎淀粉樣變分型不明確的患者,經(jīng)LMD/MS診斷出4例AL型淀粉樣變,1例Apo AIV型淀粉樣變,1例AA型淀粉樣變及1例AH型淀粉樣變;AA型淀粉樣變患者淀粉樣物質中包含SAA1及SAA2蛋白,未發(fā)現(xiàn)兩種蛋白的氨基酸序列變異。研究結論:本研究利用LMD/MS技術,對新鮮冰凍腎組織顯微切割獲取的腎小球及腎髓質組織進行蛋白質組學及生物信息學分析,豐富了腎小球蛋白質表達譜數(shù)據(jù)庫,并首次建立了正常人類腎髓質蛋白質組表達譜。免疫組化染色及LMD/MS分析發(fā)現(xiàn),對于免疫熒光不能分型的腎AL型淀粉樣變病例,采用免疫組化的方法可改進分型診斷的水平。LMD/MS技術對于部分免疫化學方法不能分型的腎AL型淀粉樣變病例可提供分型診斷,該技術能鑒定到淀粉樣纖維的多種蛋白成分,也適用于少見類型的淀粉樣變性的分型診斷并確證免疫熒光或免疫組化結果,具有一定的臨床應用前景。
[Abstract]:Research background: proteomics mass spectrometry has become one of the methods to explore the pathogenesis of kidney disease and some difficult diagnosis of kidney disease. Based on the current using laser microdissection combined with mass spectrometry (Laser microdissection and mass spectromy, LMD/MS) on human renal small ball protein expression studies were limited, learn expression study is published no renal medulla of protein group. Using LMD/MS technology can not only obtain the glomerular and renal medullary tissue to construct protein expression profile, but also on the renal lesion area is analyzed. In recent years, the use of LMD/MS technology to find some new amyloidosis subtypes, the amyloid type diagnostic level increased; at the same time the amyloidogenic protein amino acid sequence analysis is likely to find the variation of amyloidogenic proteins. Methods: in the first part, I We collected 6 cases of adjacent kidney glomerulus and renal medullary tissue obtained by LMD/MS technology and construct the proteome expression profile; bioinformatics analysis identified two sites of protein. In the second part, we analyzed the past 5 years in 45 cases of AL amyloidosis patients with clinical laboratory. Check and renal biopsy results from immunofluorescence in 20 cases of renal tissue typing is not clear with immunohistochemistry and LMD/MS classification, evaluation of immunohistochemistry and immunofluorescence typing in LMD/MS is not clear diagnosis of AL amyloidosis patients. In the third part, on 9 cases. LMD/MS analysis of patient examination and kidney tissue laboratory immunohistochemical examination were not types of difficult renal amyloid, renal amyloidosis in order to achieve the complete classification, and the use of biological information for AA amyloidosis cases methodology S Analyze the variation of amino acid sequence of AA protein. Results: in the first part, glomerular and renal medulla were identified to 1373 and 1673 proteins; and a comparison with former studies, this study identified 472 separate glomerular protein, established the normal human renal medullary proteome expression profile. Bioinformatics analysis showed glomerular protein cytoskeletal protein, renal medulla protein involved in cell metabolism, with enzymatic functions. In the second part, we found that using immunohistochemical method of immunofluorescence typing can not be part of AL amyloidosis in the differential diagnosis, the sensitivity is about 55%; the immunohistochemical method not classified patients with AL amyloidosis, LMD/MS technology can be classified diagnosis; analysis of 20 cases of LMD/MS patients, 18 cases of definite diagnosis of type, the sensitivity of 90%. in the third part, 9 cases of difficult renal amyloidosis Typing is not clear in patients after LMD/MS diagnosed 4 cases of AL amyloidosis, 1 cases of Apo AIV amyloidosis, 1 cases of AA amyloidosis and 1 cases of AH amyloidosis; AA amyloidosis include SAA1 and SAA2 protein in patients with amyloid, found no amino acid sequence two kinds of protein variation. Conclusion: in this study, using LMD/MS technology, proteomics and bioinformatics analysis of glomerular and renal medullary tissues of fresh frozen kidney tissue microdissection to obtain, enrich the glomerular protein expression database, established the normal human renal medullary proteome expression profile. Immunohistochemistry LMD/MS staining and immunofluorescence analysis showed that, for not classified renal AL amyloidosis cases by immunohistochemical method can improve renal AL type starch levels.LMD/MS typing typing for not part of immunochemical method Variant cases can provide typing diagnosis. This technology can identify various protein components of amyloid fibrils, and also can be applied to the typing diagnosis of rare types of amyloidosis and confirm the results of immunofluorescence or immunohistochemistry. It has certain clinical application prospects.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R692

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