非标记定量蛋白质组技术筛选早期糖尿病肾病大鼠的尿蛋白标志物  

Screening of urinary protein markers for early diabetic nephropathy in rats by labeled-free quantitative proteomics technique

作　　者：覃保瑜[1] 刘红[1] 黄鸿[1] 姚彦冰[1] 方丹[1] 夏宁 QIN Bao-yu;LIU Hong;HUANG Hong;YAO Yan-bing;FANG Dan;XIA Ning(Department of Geriatrics,Endocrinology and Metabolism,the First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China;Health Committee of Guangxi Zhuang Autonomous Region,Nanning 530021,China)

机构地区：[1]广西医科大学第一附院老年病学内分泌代谢科,南宁市530021 [2]广西壮族自治区卫生健康委员会,南宁市530021

出　　处：《广西医学》2019年第16期2094-2098,2104,共6页Guangxi Medical Journal

基　　金：广西医疗卫生适宜技术研究与开发课题(S201304-02)

摘　　要：目的 利用非标记定量蛋白质组技术筛选早期糖尿病肾病(DN)大鼠的尿蛋白标志物。方法 将16只大鼠随机均分为正常对照组和糖尿病模型组,采用链脲佐菌素建立糖尿病模型。将糖尿病大鼠成模前、成模后1周、成模后24周分别设为非糖尿病(ND)期、正常白蛋白尿(NA)期、微量白蛋白尿(MA)期,收集各期尿液标本。将尿标本超滤离心和浓缩后,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳粗分离尿样品的蛋白,行液相色谱-电喷雾电离串联质谱,以非标记定量蛋白质组技术筛选出差异表达的尿蛋白。采用酶联免疫吸附法对感兴趣的尿蛋白作确认。对比MA期糖尿病大鼠和正常对照组大鼠的光镜下肾组织结构。结果 与NA期比较,MA期表达显著上调的蛋白质共15个,表达显著下调的蛋白质共12个。选取尿胎球蛋白A进行确认,糖尿病大鼠MA期的尿胎球蛋白A/尿肌酐水平均高于NA期、ND期(均P<0.05)。光镜下MA期大鼠肾组织呈早期DN病理改变。结论 应用非标记定量蛋白质组技术可筛选出早期DN大鼠的差异表达尿蛋白,其中尿胎球蛋白A或可作为早期DN的诊断标志物。Objective To screen urinary protein markers for early diabetic nephropathy(DN) in rats by labeled-free quantitative proteomics technique.Methods Sixteen rats were randomly and equally divided into normal control group and diabetic model group,and the rat model of diabetics mellitus was established using streptozotocin.Among the diabetic rats,pre-modeling,one week after modeling and 24 weeks after modeling were defined as non diabetes(ND) stage,normal albuminuria(NA) stage and microalbuminuria(MA) stage,and the urine samples were collected at each stage.Crude protein was isolated from urine samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after ultrafiltration,centrifugation and concentration of urine samples,then liquid chromatography-electrospray ionization-mass spectrometry was conducted,and the differentially expressed proteins were screened out by labeled-free quantitative proteomics technique.The urinary protein of interest was confirmed by enzyme-linked immunosorbent assay.The renal tissue structure under light microscope was compared between the diabetic rats in MA stage and the rats in the normal control group.Results Compared with those in NA stage,the expression of 15 proteins was significantly up-regulated and the expression of 12 proteins was significantly down-regulated in MA stage.Urinary fetuin A was selected for confirmation,and the urinary fetuin A/urinary creatinine level of diabetic rats in MA stage was higher than that in NA stage or ND stage(all P<0.05).Under light microscope,the renal tissue of MA rats showed pathological changes of early DN.Conclusion Differentially expressed urinary protein can be screened out by labeled-free quantitative proteomics technique in rats with early DN,and urinary fetuin A may serve as a diagnostic marker for early DN.

关 键 词：糖尿病肾病 早期 尿液 蛋白质组学 尿蛋白 标志物 胎球蛋白A 大鼠 

分 类 号：R587.24[医药卫生—内分泌]