胎脑与成年脑皮质组织的蛋白质组学分析

目的 应用蛋白质组技术研究成年和胚胎脑皮质蛋白质组表达差异,初步探索在胎脑与成年人中脑蛋白的变化情况.方法 收集成人无病变区正常脑皮质1份和水囊引产胎脑组织脑皮质2份(3月胎脑组织和5月胎脑组织),提取总蛋白,通过双相电泳分离蛋白,每组样品均重复电泳,使用PDQuest7.0图像分析软件分析电泳图谱,得到具有表达差异的蛋白质,分别选取在大脑发育过程中逐步降低和逐步升高的差异点及在胎脑成脑蛋白表达中差异两倍以上的蛋白点,应用胶内酶切和基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization-time of flight-time of flight,MALDI-TOF-TOF)进行鉴定,得到肽指纹图谱,并查阅蛋白质数据库得到蛋白质的相关信息.结果 (1)成人脑组织、3月胎脑组织和5月胎脑组织的脑组织蛋白图谱分别测得642、511和527个点,图象匹配率达87%.成人脑蛋白图谱中碱性蛋白点明显多于胎脑的;(2)成人脑组织、3月胎脑组织和5月胎脑组织的脑组织单独特有蛋白分别为:172、171和152个;(3)与成人脑组织比较,3月胎脑组织和5月胎脑组织的差异蛋白质分别为131和115个;差异在2倍以上的蛋白点为60和40个,其中低表达的蛋白点分别有24和17个,高表达蛋白点为36和23个;(4)白蛋白、磷酸丙糖异构酶等8种蛋白表达均有不同的变化.脂肪酸结合蛋白7和未命名蛋白只在3月胎脑高表达,而5月胎脑和成人组均无表达;核酮糖1,5-二磷酸羧化酶/加氧酶和转导素只在成人脑中高表达,胎脑无表达.血清白蛋白却随着大脑发育逐渐降低.ATP合成酶、线粒体F0复合体亚基、磷酸丙糖异构酶随着大脑发育而逐渐升高.结论 人类从胚胎期到成人发育过程中脑部蛋白质可能会发生明显改变,这些差异蛋白的发现为研究脑发育机理提供了极其有益的线索.

Abstract：

Objective To study the differences of protein expression levels in the brain cortex of human fetus and adult with proteomics technique, and provide preliminary data on the change of proteins during brain development. Methods Proteins extracted from human temporal lobes in fetal (3 month and 5 month respectively) and adult (30 years old) brain were separated by two-dimensional gel electrophoresis (2DE).The proteins were then stained with colloidal Coomassie blue to produce a high-resolution map of the proteiome.The differential protein spots were analyzed by PDQuest 7.0 software and 8 spots,which were gradually reduced or gradually increased in brain development process and the protein spots of difference over two-fold in the brain,were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF).Results (1) On average, 642, 511 and 527 protein spots could be obtained in the temporal lobes of adult, 3 month and 5 month fetus. The matching rate of images was 87%. The basic proteins in adult brain were obviously much more than that in the fetus; (2) There were 172, 171 and 152 singular protein spots in temporal lobes of adult, 3 month and 5 month fetus respectively. (3) Compared with adult, there were 131 and 115 different protein spots in the 3 month and 5 month fetus respectively. There were 60 and 40 protein spots with more than 2 fold difference, among which 24 and 17 were down-regulated, and 36 and 23 were up-regulated respectively. (4) There was different expression in proteins such as serum albumin, triosephosphate isomerase, etc. in the 3 groups. Fatty acid binding protein 7 and unnamed proteins were only highly expressed in the 3 month brain;ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and transducin beta-1 subunit were up-regulated in adult brain. Serum albumin decreases gradually with brain development. However, ATP synthase, mitochondrial F0 complex, and triosephosphate isomerase increase gradually with brain development. Conclusion The proteins of human brain cortex were obviously changed from embryonic stage to adult. The differentially displayed proteins may provide further insight into the understanding of development of human brain.

Proteomic analysis of human fetal and adult brain cortex

Objective To study the differences of protein expression levels in the brain cortex of human fetus and adult with proteomics technique, and provide preliminary data on the change of proteins during brain development. Methods Proteins extracted from human temporal lobes in fetal (3 month and 5 month respectively) and adult (30 years old) brain were separated by two-dimensional gel electrophoresis (2DE).The proteins were then stained with colloidal Coomassie blue to produce a high-resolution map of the proteiome.The differential protein spots were analyzed by PDQuest 7.0 software and 8 spots,which were gradually reduced or gradually increased in brain development process and the protein spots of difference over two-fold in the brain,were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF).Results (1) On average, 642, 511 and 527 protein spots could be obtained in the temporal lobes of adult, 3 month and 5 month fetus. The matching rate of images was 87%. The basic proteins in adult brain were obviously much more than that in the fetus; (2) There were 172, 171 and 152 singular protein spots in temporal lobes of adult, 3 month and 5 month fetus respectively. (3) Compared with adult, there were 131 and 115 different protein spots in the 3 month and 5 month fetus respectively. There were 60 and 40 protein spots with more than 2 fold difference, among which 24 and 17 were down-regulated, and 36 and 23 were up-regulated respectively. (4) There was different expression in proteins such as serum albumin, triosephosphate isomerase, etc. in the 3 groups. Fatty acid binding protein 7 and unnamed proteins were only highly expressed in the 3 month brain;ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and transducin beta-1 subunit were up-regulated in adult brain. Serum albumin decreases gradually with brain development. However, ATP synthase, mitochondrial F0 complex, and triosephosphate isomerase increase gradually with brain development. Conclusion The proteins of human brain cortex were obviously changed from embryonic stage to adult. The differentially displayed proteins may provide further insight into the understanding of development of human brain.