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