【作者】 刘伟；

【导师】 王共先；

【作者基本信息】 南昌大学 ， 外科学， 2015， 硕士

【摘要】 目的:初步探讨不同肾结石内的蛋白质组成差异,为后续进一步探索结石内蛋白质在结石形成中的作用及其相关分子机制奠定基础。方法:收集2013年6月至2014年12月我院通过输尿管镜、经皮肾镜和腹腔镜等手术方式取石术后临床上尿路结石标本共150例。经三蒸水清洗20次、超净工作台晾干结石、充分碾磨结石标本后,取微量(约500ug)结石粉末,采用结石红外光谱分析仪分析结石组成成分;同时取1g结石粉末,加5ml RIPA蛋白裂解液溶解蛋白,经冷冻干燥浓缩蛋白,通过凝胶电泳技术对样品蛋白质进行分离及纯化,并采用考马斯亮蓝染色鉴别蛋白分子量大小;通过胶内酶解后再抽提出肽段,经液相色谱电离串联质谱(LC-ESI-MS/MS)技术鉴定蛋白质成份,并对质谱结果进行GO、Pathway初步的生物信息学分析。结果:1、在150例上尿路结石中,其中草酸钙结石33例,尿酸结石12例,草酸钙合并碳酸钙结石66例,草酸钙合并尿酸结石19例,草酸钙合并磷酸钙20例。2、SDS-PAGE电泳结果表:33例草酸钙结石内蛋白有3至4条蛋白带,主要集中70-100KD,17-26 KD;12尿酸结石内的蛋白质有5-6条蛋白带,主要集中70-100KD,30-40 KD,17-26 KD。3、蛋白质谱结果:草酸钙结石内有88种蛋白,尿酸结石内有283种蛋白;GO分析、pathway结果表明:结石内以结合功蛋白为主,占草酸钙结石蛋白总数的45.8%,占尿酸结石蛋白总数的44.34%。其中以S100A8、S100A9、纤维连接蛋白、角蛋白等炎症相关蛋白最具功能研究意义。结论:1、不同结石成份,结石内蛋白质组成及含量不同。2、相同结石成份,结石内蛋白质组成及含量略有差异。3、草酸钙结石内有88种蛋白,尿酸结石内含有283种蛋白更多还原

【Abstract】 Objectives:To Explore the different composition of proteins in the urinary kidney stones, for the subsequent further exploration within the protein’s role in stone formation and associated molecular mechanism. Methods:Firstly,we collected 150 clinical stone between June 2013 and December 2014 in our hospital about 0.5cm-4.8cm in large after surgery of percutaneous nephrolithotomy, ureteroscopy and laparoscopic, etc. After washing, drying, grinding,stones was analyzed by infrared spectroscopy. While using RIPA lysis buffer to dissolve stones, protein sample was concentrated after freezing drying, then the protein samples were isolated and purified, by gel electrophoresis The obtained proteins were analyzed with reverse-phase liquid chromatography–tandem MS. The acquired data were searched against a Swiss Prot human protein database using Matrix Science, Mascot. The identified proteins were submitted to the Ami GO Web site for gene ontology analysis. They were also sumitted to Metacore software and Kyoto Encyclopedia of Genes and Genomes website(KEGG) for pathway analysis. Results:1. In the 150 cases of urinary tract stones, there are 33 pure calcium oxalate stones, 12 uric acid stones, 66 cases of calcium oxalate stones with Calcium Carbonate, 19 cases of calcium oxalate combined with uric acid stones and 20 cases of calcium oxalate combined with Calcium phosphate2.SDS-PAGE electrophoresis results table: In 33 cases of calcium oxalate stones there are 3-4 protein bands, mainly in molecular 70-100 KD, 17-26 KD area; while 12 cases of uric acid stones within 5-6 protein bands, mainly in 70-100 KD, 30-40 KD, 17-26 KD area.3. Protein MS Results: There are 88 kinds of protein in the calcium oxalate stone, and 283 kinds of protein in the uric acid stones. GO analysis, pathway results show that: binding proteins in the stones, accounting for 45.8% of the total protein in the calcium oxalate stone, while 44.34% in the uric acid stones. Among them, S100A8, S100A9, fibronectin, keratin and other inflammatory protein may be related to calculi forming. Conclusions:1. protein composition and content vary in different kind of stones.2. protein composition and content in the same kind of stones vary slightly3. There are 88 kinds of protein in the calcium oxalate stone, 283 kinds of protein in the uric acid stone.更多还原