应用iTRAQ技术筛选儿童皮质发育障碍性癫痫差异表达蛋白  

Application of iTRAQ in screening differentially expressed proteins in children with cortical dysplasia-induced epilepsy

作　　者：覃璐[1] 杨轶轩[2] 晏宁[3] 陈阳美[1] 陈莉芬[1] 

机构地区：[1]重庆医科大学附属第二医院神经内科,重庆400010 [2]重庆医科大学附属第二医院感染科,重庆400010 [3]重庆医科大学附属大学城医院神经内科,重庆401331

出　　处：《第三军医大学学报》2015年第14期1400-1405,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81171225)~~

摘　　要：目的筛选、鉴定儿童皮质发育障碍性癫痫的差异表达蛋白。方法以儿童皮质发育障碍性癫痫脑组织、脑外伤术后的儿童正常脑组织(对照组)为对象,采用同位素相对标记与绝对定量技术(i TRAQ)、双向液相色谱与串联质谱(2D-LC-MS/MS)鉴定儿童皮质发育障碍性癫痫组织中差异表达蛋白,对所获得差异蛋白进行生物信息学分析,筛选出具有潜在研究价值的候选蛋白,最后应用实时定量PCR和Western blot验证这些候选蛋白在儿童皮质发育障碍性癫痫中的表达。结果共鉴定出153种差异表达蛋白(64种蛋白上调,89种蛋白下调)。Gene ontology(GO)功能分析提示这些差异性蛋白主要参与酶催化活性的调节,蛋白质功能相互作用网状图发现转铁蛋白(transferrin,TF)、载脂蛋白A1(apolipoprotein A1,APOA1)、α2-巨球蛋白(alpha-2-macroglobulin,A2M)、三磷酸腺苷酶(ATPase,ATP6V1A)、波形蛋白(vimentin,VIM)处于网状图的交叉点。运用实时定量PCR及Western blot验证,发现这5种蛋白质mRNA及蛋白表达水平与质谱结果一致(P<0.05)。结论采用i TRAQ结合2DLC-MS/MS技术,能有效地筛选出儿童皮质发育障碍性癫痫中差异表达的蛋白。Objective To screen the differentially expressed proteins in children with disorders of cortical development（ DCD） accompanied by epilepsy. Methods The brain tissues from children with DCDinduced epilepsy and normal children（ control group） operated for head trauma were selected as specimens.Isobaric tags for relative and absolute quantitation（ i TRAQ） technique and 2-dimensional liquid chromatography-tandem mass spectrometry（ 2D-LC-MS/MS） were used for identifying the differentially expressed proteins. The differentially expressed proteins were analyzed by bioinformatics for searching candidate proteins. Finally,the candidate proteins were verified by real-time PCR and Western blotting.Results A total of 153 proteins showed significantly different expression,and included 64 up-regulated proteins and 89 down-regulated proteins. Gene ontology（ GO） functional analysis showed that the differentially expressed proteins were mainly involved in catalytic activity. The protein interaction network showed that transferrin（ TF）, apolipoprotein A1（ APOA1）, alpha-2-macroglobulin（ A2M）, ATPase（ ATP6V1A）,and vimentin（ VIM） were located at the crossed nodes. The expression levels of TF,APOA1,A2 M,ATP6V1A and VIM were verified by real-time PCR and Western blotting,and the results were consistent with those of the quantitative mass spectrometry（ P〈0. 05）. Conclusion Differentially expressed proteins in the children with DCD-induced epilepsy can be effectively screened by i TRAQ technique combined with 2D-LC-MS/MS.

关 键 词：皮质发育障碍 癫痫 蛋白质组学 ITRAQ 儿童 

分 类 号：R341[医药卫生—基础医学] R394-33