大鼠肠缺血再灌注损伤后肠黏膜差异表达蛋白质组的分离与鉴定  

Separation and identification of differential expression proteome of intestinal mucosa after intestinal ischemia/reperfusion injury in rats

作　　者：李云胜[1] 刘克玄[1] 刘家欣[1] 李毅[1] 黄文起[1] 

机构地区：[1]中山大学附属第一医院麻醉科,广州510080

出　　处：《中华生物医学工程杂志》2009年第3期170-175,共6页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金（30672021）

摘　　要：目的研究大鼠肠缺血再灌注（U／R）损伤后肠黏膜差异表达蛋白质的变化，为探讨II／R损伤后肠黏膜损伤的分子机制提供理论依据。方法16只SD大鼠，完全随机法分为假手术组（S组）和II／R损伤组（II／R组），每组8只。S组只分离肠系膜上动脉（SMA）但不阻断，II／R组阻断SMA 1h后再开放1h。再灌注结束即刻刮取肠黏膜，利用高分辨双向凝胶电泳对肠黏膜组织进行蛋白质分离，Image Master 2D Elite5．0图像分析软件进行分析。应用基质辅助激光解析电离飞行时间质谱获取肽质量指纹图谱，检索NCBInr蛋白数据库鉴定表达差异的蛋白质，明确其生物学功能。结果S组和II／R组分别有（1287±20）和（1404±20）个蛋白质点得到分离，组内匹配点数分别有（1136±20）和（1197±20）个，匹配率分别为88．3％和85.3％；组间匹配点数为（1084±20）个，匹配率为84．2％。发现16个表达差异的蛋白质点，其中12个在II／R组中表达下调，4个表达上调。通过质谱分析鉴定13个蛋白质，检索NCBInr数据库查询到其主要是顺乌头酸酶、丙酮酸激酶和细胞色素C还原酶，蛋白二硫化物异构酶A3、醛脱氢酶和醛糖还原酶，前3种酶与改善能量代谢相关，后3种酶与抗氧化应激、抑制凋亡相关。结论建立了重复性较好的正常与II／R损伤后大鼠肠黏膜组织的双向凝胶电泳图谱。II／R的损伤机制可能与顺乌头酸酶、丙酮酸激酶、细胞色素C还原酶、蛋白二硫化物异构酶A3、醛脱氢酶和醛糖还原酶的下调有关。Objective To investigate the changes of differential expression protein in rats intestinal mucosa after intestinal ischemia/reperfusion （II/R） injury and provide theory evidence for the mechanisms exploration of intestinal mucosa injury after II/R injury at molecular level. Methods Sixteen SD rats were randomly divided into sham operation group （S group, n=8） and II/R injury group （II/R group, n=8）. In the S group, superior mesenteric artery （SMA） was only separated without occlusion. In the II/R group, ischemia/reperfusion was preformed by SMA occlusion for 1 hour and reperfusion for 1 hour. The intestinal mucosa of rats was scratched immediately after reperfusion and total proteins in intestinal mucosa tissue were separated by immobilized pH gradient （IPG） based high resolution two-dimensional gel electrophoresis. The proteins with differential expression were analyzed using Image Master 2D Elite 5.0 image analysis software. Peptide mass finger-printing （PMF） was obtained by matrix assisted laser desorption ionization-time of flightmass spectrum （MALDI- TOF-MS）. The differential expression proteins and its biological functions were identified by searching NCBInr proteins database. Results Image analysis revealed that average of 1287±20 and 1404 ± 20 protein spots were separated in intestinal mucosal tissue of two groups respectively. The matched spots were 1136±20 and 1197±20 in intragroup, and the matching rates were 88.3% and 85.3% respectively. There were 1084±20 matched protein spots between two groups, and the matching rate was 84.2%. Sixteen differential expression protein spots were found, of which 12 were down-regulated and 4 were up-regulated in the II/R group. Thirteen proteins were identified by mass spectrometry. In 13 proteins, cytoplasmic cis- aeonitate hydratase, pyruvate kinase and cytoehrome C reduetase were related to the improvelnent of energy metabolism, and protein disulfide-isomerase A3 , aldehyde dehydrogenase and aldose reductase were related to anti- oxi

关 键 词：缺血再灌注损伤 蛋白质组学 肠黏膜 电泳 凝胶 双向 光谱分析 质量 

分 类 号：R285.5[医药卫生—中药学]