缺血预处理抗大鼠肠缺血再灌注肠黏膜损伤的蛋白质组学研究

目的采用蛋白质组研究技术分离、鉴定缺血预处理（IPC）抗大鼠肠缺血再灌注（Ⅱ／R）肠黏膜损伤相关蛋白，探讨其肠保护分子机制。方法将16只SD大鼠，随机分为Ⅱ／R组和IPC组。Ⅱ／R组阻断肠系膜上动脉60min后再开放60min;IPC组在阻断肠系膜上动脉前先阻断20min后再开放5min。余同Ⅱ／R组。再灌注结束即刻刮取肠黏膜，利用高分辨双向电泳对肠黏膜组织进行蛋白质分离．Image Master 2D Elite 5．0图像软件进行分析。应用基质辅助电离解析飞行时间质谱获取肽质量指纹图谱．检索数据库鉴定表达差异的蛋白质，明确其生物学功能。结果双向电泳发现，Ⅱ／R组及IPC组分别有蛋白质点（1404±20）个和（1338±20）个。10个点进行质谱分析，8个蛋白质点经过检索与已知蛋白质匹配．这些蛋白功能涉及到抗氧化、抑制凋亡及改善能量代谢。RT-PCR分析提示IPC上调醛糖还原酶的表达。Western blot分析提示IPC上调醛脱氢酶的表达。结论比较蛋白组学研究揭示IPC对肠缺血再灌注损伤的保护机制可能与其上调了一些具有抗氧化、抑制细胞凋亡及改善能量代谢作用的蛋白有关。

Proteomics study of intestinal mucosa after ischemic preconditioning against intestinal ischemic reperfusion injury in rats

Objective To identify associated proteins involved in the molecular response of ischemic preconditioning (IPC) against intestinal ischemia/reperfusion(Ⅱ/R) in the intestinal mucosa of rats. Methods Sixteen SD rats were randomly divided into Ⅱ/R and IPC groups. Ⅱ/R injury in rats was produced by clamping superior mesenteric artery for 60 min followed by 60 min reperfusion. IPC was elicited by 20 min ischemia and 5 min reperfusion before index ischemia. The intestinal mucosa was scratched immediately after 60 min of reperfusion and total proteins were separated by immobilized pH gradient(IPG) based on two-dimensional gel electropboresis(2-DE). The differentially expressed proteins were analyzed using Image Master 2D Elite 5.0 image analysis software and identified by MALDI-TOF-MS. The biological information of these proteins was searched in the database of these peptide mass finger-printing (PMF). Western blotting and RT-PCR were used to validate the differentially expressed proteins. Results Image analysis revealed that averages of 1404±20 and 1338±20 were detected in Ⅱ/R and IPC groups. A total of 10 spots yielded good spectra, and 8 spots matched with known proteins after database searching. These proteins were mainly involved in anti-oxidation, inhibiting apoptosis and energy metabolism. Western blot confirmed up-regulation of aldehyde dehydrogenase and RT-PCR confirmed up-regulation of aldose reductnse in IPC group. Conclusion The clues provided by comparative proteome strategy will shed light on molecular mechanisms of IPC against Ⅱ/R injury.