泌尿系草酸钙结石蛋白分析  

Analysis of Protein Ingredient in Urine Calcium Oxalate Stones

作　　者：叶韬[1,2] 王义兵 刘伟[2] 罗晓明[4] 王共先[2,5] 黄学明[5] 刘伟鹏 习海波 余月[2] 傅斌[2,5] YE Tao;WANG Yibing;LIU Wei(.Department of Urologic Surgery,Affiliated medical school of Nanchang University(First People’s Hospital of Jiujiang),Jiujiang,Jiangxi 332000,China;Department of Emergency,The Second Affiliated Hospital of Nanchang University,Nanchang,Jiangxi 330006,China;Department of Pharmacy,Jiangxi Maternal And Child Health Hospital,Nanchang,Jiangxi 330006,China;Department of Urologic Surgery,The First Affiliated Hospital of Nanchang University,Nanchang,Jiangxi 331000,China;Jiangxi Institute of Urinary Surgery,Nanchang,330006,China)

机构地区：[1]江西省九江市第一人民医院泌尿外科,九江332000 [2]南昌大学第一附属医院泌尿外科,南昌330006 [3]南昌大学第二附属医院急诊科,南昌330006 [4]江西省妇幼保健院药剂科,南昌330006 [5]江西省泌尿外科研究所,南昌330006

出　　处：《江西医药》2018年第3期205-208,共4页Jiangxi Medical Journal

基　　金：江西省卫计委科技计划20152099;国家十二五科技支撑计划2013BAI05B00

摘　　要：目的研究泌尿系草酸钙结石蛋白成分特点,为后续进一步探索蛋白质在结石形成中的作用机制奠定基础。方法随机收集临床结石样本20例,采用结石红外光谱分析仪分析结石组成成分;收集对应患者尿液进行尿液分析及细菌培养。采用RIPA蛋白裂解液溶解结石内蛋白成分,通过冷冻干燥浓缩提取结石内蛋白。采用细菌蛋白质提取试剂盒提取大肠杆菌蛋白。应用Bradford法测定蛋白浓度,再通过SDS-SPAGE电泳纯化蛋白,银染染色和考马斯亮蓝染色分别鉴定结石和大肠杆菌内蛋白分子量大小,进行蛋白比对。通过胶内酶解后抽提肽段,经液相色谱仪和质谱仪鉴定蛋白质成份,对质谱结果进行Gene Ontology等生物功能信息分析。结果⑴草酸钙结石平均蛋白浓度为0.57μg/μL。存在泌尿道感染时的草酸钙结石内蛋白含量相对较高(P<0.05)。大肠杆菌蛋白浓度为5.38μg/μL。⑵SDS-PAGE电泳结果显示草酸钙结石内蛋白有3至4条蛋白带,主要集中在25.0-35.0KDa和66.2-116.0KDa;大肠杆菌的蛋白条带主要集中在35.0KDa-116.0KDa。蛋白条带图谱提示草酸钙结石与大肠杆菌内蛋白质成份具有重叠性。⑶蛋白质谱分析显示草酸钙结石内有88种蛋白成分,以结合功能蛋白为主;其中包括S100A8、S100A9、纤维连接蛋白、角蛋白等炎症相关蛋白。结论泌尿系草酸钙结石内蛋白成分复杂,不仅与大肠杆菌蛋白成分存在交叉,而且有炎症相关蛋白,提示草酸钙结石形成与尿路感染存在关联,结石内炎症性蛋白成分在结石形成过程中的作用有待深入研究。Objective To Explore the characteristics of Protein Ingredient in Urine Calcium Oxalate Stones,for the subsequent further exploration within the protein's role in stone formation and associated molecular mechanism.Methods 20 clinical stone were collected after surgery.Stones were analyzed by infrared spectroscopy to determine the components of stones.RIPA lysis buffer were used to dissolve stones,and protein sample was concentrated after freezing drying.Qproteome Bacterial Protein Prep Kit were adopted to extract e.coli protein.Then we measured the protein concentration using Bradford method,and isolated and purified by gel electrophoresis.The molecular weight of the protein was identified by using coomassie brilliant blue staining and silver staining,and compared the protein band patterns.The obtained proteins were analyzed with reverse-phase liquid chromatography-tandem mass spectrometry.The acquired data were searched against a Swiss Prot human protein database using Matrix Science,Mascot.The identified proteins were submitted to the AmiGO Web site for gene ontology analysis.They were also sumitted to Metacore software and Kyoto Encyclopedia of Genes and Genomes website(KEGG)for analysis.Results⑴The average protein concentration of calcium oxalate stone was 0.57μg/μL.There was a relatively high protein content of calcium oxalate in urinary tract infection(P<0.05).E.coli protein concentration was 5.38ug/uL.⑵SDS-PAGE electrophoresis results indicates that there are at least 3-4 protein bands in calcium oxalate stones,mainly concentrating in molecular 66.2-116.0KD,25.0-35.0 KD.The protein bands of e.coli are mainly concentrated in 35.0KDa-116.0KDa.The protein band pattern indicated that the protein composition of calcium oxalate stone and e.coli were overlapping.⑶Protein Mass Spectrometry showed that there are 88 kinds of protein in the calcium oxalate stone,which were mainly binding proteins.Among them,S100A8,S100A9,fibronectin,keratin and etc,which may be associated with inflammation.Conclusion The

关 键 词：草酸钙结石 尿路感染 大肠杆菌 蛋白分析 

分 类 号：R691.4[医药卫生—泌尿科学]