正常肾组织蛋白质组学分析及淀粉样变性质谱诊断分型研究

发布时间：2018-04-18 13:55

本文选题：肾小球 + 肾髓质　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究背景:基于质谱的蛋白质组学技术已成为探索肾脏疾病发病机制及某些疑难肾脏疾病诊断的方法之一。目前利用激光显微切割联合质谱(Laser microdissection and mass spectromy,LMD/MS)技术对人肾小球进行蛋白质表达谱的研究有限,对肾髓质进行蛋白质组学表达谱的研究尚未见发表。采用LMD/MS技术不仅可分别获取肾小球及肾髓质组织以构建蛋白表达谱,也可对肾病变组织区域进行分析。近年来,利用LMD/MS技术发现一些新的淀粉样变亚型,使得淀粉样变分型诊断水平有所提高;同时对致淀粉样变蛋白进行氨基酸序列分析有可能发现变异的致淀粉样变蛋白。研究方法:在第一部分工作中,我们收集6例癌旁肾组织,通过LMD/MS技术获取肾小球及肾髓质组织并构建蛋白质组表达谱;对两个部位所鉴定到的蛋白进行生物信息学分析。在第二部分工作中,我们分析了近5年45例AL型淀粉样变性病患者临床、实验室检查及肾活检病理结果,选取20例肾组织免疫荧光分型不明确的患者进行免疫组化及LMD/MS分型,评价免疫组化及LMD/MS在免疫荧光分型不明确的AL淀粉样变患者中的诊断意义。在第三部分工作中,对9例临床、实验室检查及肾组织免疫化学检查均不能分型的疑难肾淀粉样变患者进行LMD/MS分析,以期达到肾淀粉样变的完整分型诊断,并利用生物信息学方法对AA型淀粉样变病例的SAA蛋白进行变异氨基酸序列分析。研究结果:第一部分中,肾小球及肾髓质分别鉴定到1373及1673种蛋白;与以往研究比较,本研究单独鉴定到472种肾小球蛋白,并首次建立了正常人类肾髓质蛋白质组表达谱。生物信息学分析显示肾小球蛋白多为细胞骨架蛋白,肾髓质蛋白多参与细胞代谢、具有酶解功能。第二部分中,我们发现应用免疫组化方法可对免疫荧光不能分型的部分AL型淀粉样变进行分型诊断,敏感性约为55%;对于部分免疫组化方法不能分型的AL型淀粉样变患者,LMD/MS技术仍可进行分型诊断;20例行LMD/MS分析的患者中,18例可明确分型诊断,敏感性为90%。第三部分中,9例疑难肾淀粉样变分型不明确的患者,经LMD/MS诊断出4例AL型淀粉样变,1例Apo AIV型淀粉样变,1例AA型淀粉样变及1例AH型淀粉样变;AA型淀粉样变患者淀粉样物质中包含SAA1及SAA2蛋白,未发现两种蛋白的氨基酸序列变异。研究结论:本研究利用LMD/MS技术,对新鲜冰冻肾组织显微切割获取的肾小球及肾髓质组织进行蛋白质组学及生物信息学分析,丰富了肾小球蛋白质表达谱数据库,并首次建立了正常人类肾髓质蛋白质组表达谱。免疫组化染色及LMD/MS分析发现,对于免疫荧光不能分型的肾AL型淀粉样变病例,采用免疫组化的方法可改进分型诊断的水平。LMD/MS技术对于部分免疫化学方法不能分型的肾AL型淀粉样变病例可提供分型诊断,该技术能鉴定到淀粉样纤维的多种蛋白成分,也适用于少见类型的淀粉样变性的分型诊断并确证免疫荧光或免疫组化结果,具有一定的临床应用前景。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R692

【相似文献】