【作者】 陈玉强；

【导师】 房学东；

【作者基本信息】 吉林大学 ， 外科学， 2011， 博士

【摘要】 肠道缺血再灌注可导致全身炎症反应综合征和多器官功能障碍综合征的发生。但肠道缺血再灌注所引起多器官功能障碍综合征例如肝、肺、肾等的发生机制至今仍尚未明确。缺血再灌注后产生大量氧自由基以及由其所引发的中性粒细胞激活、炎性介质的生成(如TNF-α)以及蛋白酶增多等导致一系列的连锁反应。生成的TNF-α可以使中性粒细胞聚集粘附于内皮细胞,引起肺损伤。其在作用于内皮细胞时又可激活Caspase-8,二者共同作用进一步加重肺内皮细胞损伤。而在再灌注期间大量无活性的NF-κB被激活,活化后的NF-κB正反馈调节于TNF-α,使TNF-α基因转录增强,数量增多,二者协同促使机体产生更多的氧自由基及炎性介质,其代谢产物大量堆积在肺泡内皮细胞上,导致肺组织结构改变和功能障碍,最终出现肺功能衰竭。目的：观察TNF-α诱导的Caspase-8和NF-KB在大鼠小肠缺血再灌注后肺上皮细胞中的表达。方法：健康雄性Wistar大鼠48只,清洁级,2-3月龄,体重200-250 g,随机分为正常组、假手术组、对照组和实验组,建立小肠缺血再灌注模型,于缺血1h后腹腔注射TNF-α,再灌注24h后取肺泡上皮细胞,HE染色法从组织学和形态学对肺损伤进行评价,采用免疫组织化学染色技术和Westren blottinf对Caspase-8和NF-κB的表达进行检测及定量分析。结果：Caspase-8的阳性表达位于肺泡上皮细胞胞浆,与正常组、对照组和假手术组相比,实验组中Caspase-8的表达有统计学意义(P<0.05)。NF-κB的阳性表达位于肺泡上皮细胞胞核和胞浆,与正常组、对照组和假手术组相比,实验组中NF-κB的表达有统计学意义(P<0.05)。结论：大鼠小肠缺血再灌注损伤后在TNF-α诱导的肺上皮细胞凋亡中Caspase-8与NF-κB共同参与此病理过程。肺损伤后肺组织湿干比值(W/D)升高,两组实验中实验组湿干比值(W/D)均显著高于正常组、假手术组和对照组,证明实验组大鼠肺损伤较重。我们的实验表明实验组大鼠肺组织高表达Caspase-8与NF-κB,说明二者被激活后,均参与到小肠缺血再灌注后TNF-α诱导的肺损伤中,是TNF-α细胞信号通路中的重要组成部分。抑制二者表达活性,可能减轻肠系膜血管缺血性疾病导致的急性呼吸窘迫综合征和多器官功能障碍综合征。更多还原

【Abstract】 Intestine ischemia -reperfusion can cause systemic inflame- matory responses and multiple organ dysfunction syndromes. But the mechanism of multiple organ dysfunction syndrome damage such as lungs, liver, kidney is not clear so far induced by intestine ischemia reperfusion.Generating a great deal of oxidizing free radicals after intestine ischemia-reperfusion,and more neutrophile granulocyte being activated, inflammatory mediator created for example TNF-a and proteinase growing in number,all these can give rise to a series of chain reactions。TNF-αcan make neutrophile granulocyte gather together with lung endothelial cells, and this can lead to lung injury. On the same time, TNF-a can make Caspase-8 be activated, both of them can aggravate lung injury further more. During the time of ischemia -reperfusion a number of inactive NF-κB were activated. They can make positive feedback regulation with TNF-a, enhance its transcription and increase its number. Then they can cooperate with each other to urge generating a great deal of oxidizing free radicals and inflammatory mediator. The metabolites of them were accumulated in the lung alveolar epithelial cells, leading to appear lung structural modification and dysfunction, finally lung nonfunction.Objective:to observe the expression of Caspase-8 and NF-κB acted in lung type II alveolar epithelial cells induced by TNF-alpha after rat intestinal ischemia-reperfusion.Methods:48 male Wistar rats (8-12 weeks) weighing 200-250g were randomly divided into normal group, sham group, control group and the experimental group; and then establishing small intestine ischemia -reperfusion model; extracting materials of the lung typeⅡepithelial cells at 24 hours after ischemia-reperfusion; using histotherapy and immunohistochemical staining techniques and image analysis technology, to observe histopathological changes and the expression of Caspase-8 and NF-κB.Results:The masculine Caspase-8 lies in alveolar epithelial cell cytolymph, and its expression strength in the experimental group is higher than the control group, the sham group and the normal group (P<0.05).Positive expression of NF- kB lies in alveolar epithelial cell nuclei or cytolymph and its expression strength in the experimental group is higher than the control group, the sham group and the normal group (P<0.05).Conclusion:Caspase-8 and NF-κB which took part in the process of pathological changes during the apoptosis of lung epithelial cells caused by TNF alpha after the rats’intestinal ischemia-reperfusion injury. After lung injury, the value of Wet Dry ratio (W/D) was up. Both in the two sets of experiments, the value of W/D in the experiment group show that was higher than the normal group, the sham group and the control group. All that can prove the rats of the experiment group were injured more heavily in the lung injury.Our experiments indicated that the Caspase-8 and NF-KB had a high expression in the experiment rats’lung organizes.Which explained the two after being activated, all took part in lung injury induced by TNF- alpha after rat intestinal ischemia-reperfusion. And this can indicate they would take an important part in TNF alpha cell signaling pathways. So inhibiting its expression activity may reduce acute respiratory distress syndrome and multiple organ dysfunction syndromes which were caused by the mesenteric vascular ischemic diseases. But whether Caspase-8 and NF-KB have a relationship with each other we have no idea, and this will still need a further research.更多还原