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目的:研究兔肠道部分缺血再灌注(I/R)损伤过程中外周血中性粒细胞(PMN)凋亡的变化及机制。方法:大耳白兔55只,分为肠部分 I/R 组、假手术对照组、正常对照组,用自行设计的血流阻断器阻断肠系膜上动脉(SMA)血流50%,维持4h,制作肠道部分缺血再灌注操作动物模型:采用流式细胞术(AnnexinV/PI 双染法)测定外周血 PMN 凋亡变化,采用放免法测定血浆内 IL-6和 TNF-α水平,采用免疫组织化学方法观察外周血 PMNcaspase-3蛋白的表达,同时观察脏器功能和病理形态学改变。结果:肠部分 I/R 组,在肠道缺血期血浆 IL-6T 和TNF-α水平及外周血 PMN 凋亡比例均明显升高;在肠道再灌注期至术后1d 外周血 PMN 凋亡比例骤然减少,而血浆 IL-6和 TNF-α在肠道再注期处于较高水平:外周血 PMN caspase-3蛋白在肠道缺血期呈现高表达,而在再灌注期后表达明显降低。结论:肠道 I/R 全过程中 PMN 的凋亡与多种信号启动 caspase-3蛋白的表达正相关。在肠道缺血期,IL-6及 TNF-α可能是外周血 PMN 的凋亡过程的诱导分子或促进分子:而在肠道再灌注期,外周血 PMN 凋亡锐减可能是导致 TNF-α、IL-6等细胞因子进一步增加的原因。
Objective: To investigate the changes and mechanisms of apoptosis of peripheral blood neutrophils (PMN) during intestinal ischemia / reperfusion (I / R) injury in rabbits. Methods: 55 rabbits were divided into intestinal I / R group, sham operation control group and normal control group. 50% of the superior mesenteric artery (SMA) blood flow was blocked by self designed blood flow blocker 4h, making intestinal part of ischemia-reperfusion animal model: using flow cytometry (AnnexinV / PI double staining) to detect the changes of peripheral blood PMN, using radioimmunoassay determination of plasma levels of IL-6 and TNF- Immunohistochemistry was used to observe the expression of PMNcaspase-3 protein in peripheral blood and to observe the changes of organ function and pathomorphology. Results: The levels of IL-6T and TNF-α in the intestine ischemic phase and the percentage of apoptotic PMN in peripheral blood were significantly increased in the intestinal part of I / R group. In the intestinal reperfusion period, The percentage of death was abruptly decreased, while the plasma levels of IL-6 and TNF-α were remarkably higher in the intestinal reperfusion period. Peripheral blood PMN caspase-3 protein was overexpressed in the intestinal ischemia, but was significantly increased after reperfusion reduce. Conclusion: The apoptosis of PMN in the whole process of intestinal I / R is positively correlated with the activation of caspase-3 protein by various signals. During intestinal ischemia, IL-6 and TNF-α may be the inducing molecules or promoters of PMN apoptosis in peripheral blood. In the intestinal reperfusion period, the apoptosis of peripheral PMN may be the result of TNF- α, IL-6 and other cytokines cause further increase.