CAJ | 학술논문

目的应用蛋白质组学方法筛选新生先天性甲状腺功能减退症大鼠大脑差异表达蛋白质,为阐明CH致脑发育障碍发病机制提供有价值的线索.方法制作CH仔鼠动物模型,于出生时称重后处死仔鼠,取大脑,提取大脑皮质总蛋白,Bradford法检测蛋白质浓度.应用双向电泳(2-DE)技术分析新生正常仔鼠与CH仔鼠大脑蛋白质差异表达情况.选择重复性、分辨率好且表达差异明显的蛋白质点进行质谱分析.RIA法检测各组大鼠血清FT3、FT4水平.结果新生甲减组仔鼠体重、FT3、FT4水平均低于正常组仔鼠(t体重=-8.07,tFT3=5.39,tFT4=7.62,P＜0.01).建立了较稳定的正常与CH仔鼠大脑2-DE图谱,筛选出7个重复性、分辨率好且表达差异明显的蛋白质点进行质谱分析.MALDI-TOF-MS质谱分析鉴定了相关的7个差异表达蛋白,包括塌陷反应介导蛋白2、肌动蛋白相关蛋白2/3复合物第5亚单位、泛素结合酶E2-25K、ATP合酶D亚单位、Cu-Zn超氧化物歧化酶、突触核蛋白α、核苷二磷酸激酶.结论神经元突触形成异常、ROS产生异常增多、细胞凋亡等多条途径可能参与了CH致脑发育障碍,本研究为探讨CH致脑发育障碍机制提供了重要线索.
목적 응용단백질조학방법사선신생선천성갑상선공능감퇴증대서대뇌차이표체단백질,위천명CH치뇌발육장애발병궤제제공유개치적선색.방법 제작CH자서동물모형,우출생시칭중후처사자서,취대뇌,제취대뇌피질총단백,Bradford법검측단백질농도.응용쌍향전영(2-DE)기술분석신생정상자서여CH자서대뇌단백질차이표체정황.선택중복성、분변솔호차표체차이명현적단백질점진행질보분석.RIA법검측각조대서혈청FT3、FT4수평.결과 신생갑감조자서체중、FT3、FT4수평균저우정상조자서(t체중=-8.07,tFT3=5.39,tFT4=7.62,P＜0.01).건립료교은정적정상여CH자서대뇌2-DE도보,사선출7개중복성、분변솔호차표체차이명현적단백질점진행질보분석.MALDI-TOF-MS질보분석감정료상관적7개차이표체단백,포괄탑함반응개도단백2、기동단백상관단백2/3복합물제5아단위、범소결합매E2-25K、ATP합매D아단위、Cu-Zn초양화물기화매、돌촉핵단백α、핵감이린산격매.결론 신경원돌촉형성이상、ROS산생이상증다、세포조망등다조도경가능삼여료CH치뇌발육장애,본연구위탐토CH치뇌발육장애궤제제공료중요선색.
Objective To screen differentially expressed brain proteins with proteomic method in cerebral cortex of neonatal rats with congenital hypothyroidism. Method From the 13th day of gestation,pregnant Wistar rats from the experimental group were given intragastrically with 2. 5 ml of 1%propylthiouracil daily. Cerebral cortex specimens were collected from the control and hypothyroidism neonatal rats. Two-directional electrophoresis (2-DE) was applied to analyze protein expression diversities between the euthyroid and hypothyroidism neonatal rat cerebral cortex. Protein spots with significantly different expression were screened and identified by mass spectrometry. Radioimmunoassay (RIA) was used to analyze serum FT3 , FT4 levels of each groups. Result The body weight of hypothyroid neonatal rats were lower than those in the corresponding control group (t = -8.07, P ＜0. 01 ). The FT3 levels of hypothyroid neonatal rats were lower than those in the corresponding control group ( t = 5. 39, P ＜ 0. 01 ). The FT4 levels of hypothyroid neonatal rats were lower than those in the corresponding control group (t = 7.62, P ＜ 0. 01 ).Stable 2-DE maps of normal and CH neonatal rat were constantly obtained. The maps were analyzed by software. Seven protein spots with high reproducibility, high resolution and significantly different expression were chosen and identified by mass spectrometry, including collapsing response mediator protein 2, actin related protein 2/3 complex subunit 5, ubiquitin-conjugating enzyme E2-25K, ATP synthase subunit d, CuZn superoxide dismutase, synuclein alpha, and nucleoside diphosphate kinase. Conclusion The value of this research is demonstrated here by the identification of several proteins known to be associated with nerve synapse structures formation, cell survival, metabolism, cell signal transduction, neural differentiation and nerve growth in the central nervous system. Furthermore this study identified several proteins except for collapsing response mediator protein 2 and Cu-Zn superoxide dismutase that have not previously been described in the literature and which may play an important role as either sensitive biomarkers of brain dysfunction caused by congenital hypothyroidism. In congenital hypothyroidism, brain development retardation may be related with some important processes, including abnomal synaptic formation, excess ROS production and apoptosis. The above-mentioned proteins may play critical roles in the processes, which provide valuable clues to clarify the pathogenesis of brain developmental disorders induced by congenital hypothyroidism.