【作者】 袁勇；

【导师】 陈嘉勇；

【作者基本信息】 昆明医科大学 ， 外科学， 2012， 博士

【摘要】 背景及目的：小肠缺血再灌注损伤(Ischemia Reperfusion Injury, IRI)是外科手术和创伤病人高发病率和死亡率的重要因素。例如在腹主动脉瘤、心肺旁路手术、绞窄性疝、新生儿坏死性小肠炎和小肠移植等肠血流量中断的情况下,IRI是非常常见的重要阶段,在败血症和低血容量性休克时,小肠I RI也经常发生。血流供应的中断造成局部的缺血损伤易于影响代谢活跃的组织,相矛盾的是,缺血组织血流恢复时将会启动一系列的事件,导致细胞进一步的损伤,即再灌注损伤。再灌注损伤的严重性往往超过了最初的缺血性损伤。在IRI中易于发生自由基介导的损伤,发生分子和生化等变化。因此,血液供应减少或中断以及随后的血供恢复将造成组织器官的严重损害,即缺血再灌注损伤(IRI)在内脏器官中,小肠是对IRI最敏感的器官之一。缺血时容易损伤由不稳定细胞组成的肠道,随后的血液再灌注则会进一步损伤小肠粘膜细胞。有研究认为,位于绒毛的顶端肠上皮细胞对缺血敏感性增加的原因是由于其位于中央动脉的尽头,具有较低氧分压,对局部缺血较敏感。同时,小肠也是产生各种急性期蛋白和细胞炎症因子的主要场所,小肠IRI不仅会影响小肠本身,还能影响远隔器官的功能和完整性。在缺血发生起始区域的异常改变主要是影响细胞线粒体,特别是影响三磷酸腺苷(Adenosine Triphosphate, ATP)的产生,导致细胞发生缺氧性病理改变,随着缺血缺氧的进一步加重以及随之而来的再灌注损伤,小肠将产生大量的氧自由基等有害物质,损伤微血管、释放炎性细胞因子、激活补体和活化中性粒细胞。因此,小肠IRI被认为是SIRS或MODS最主要的使动因素之一有研究表明缺血预处理(Ischemic Preconditioning, IPr)即在器官缺血前给予一次或多次短暂的缺血与再灌注处理,可减轻心、脑、小肠、肾等多种器官的IRI,但由于小肠IRI常见于肠扭转、肠系膜上动脉血栓形成、休克、器官移植术和心力衰竭等,临床上患者多在发生肠缺血后就诊,难以进行预处理,这就极大地限制了其临床应用。因此,有人研究在缺血发生后实施的缺血后处理(Ischemic Postconditioning. IPo),即在缺血后再灌注开始时立刻给予一次或多次短暂再灌注与缺血交替循环处理,可以减轻再灌注损伤,具有更好的临床应用前景。但这一新发现的内源性保护机制尚未完全阐明。本研究通过建立大鼠小肠IRI模型,研究IPo对细菌移位、小肠病理改变、小肠粘膜线粒体形态、功能进行研究,以期为临床治疗小肠IRI提供一定的理论和实验依据,并对IPo的保护性机制做进一步研究。方法：实验选用SPF级SD大鼠建立小肠IRI模型,采用IPr、IPo与空白对照组和IR组进行对比研究。第二章研究IPo对小肠细菌移位及病理损伤的保护作用：通过实验前给予标记的条件致病菌灌胃,实验通过对不同组别的肠外组织进行细菌移位检测,以及对小肠病理损伤分级评分来了解IPo对小肠粘膜机械屏障损伤的保护作用。第三章进行IPo对大鼠小肠IRI粘膜细胞线粒体保护作用的实验研究：线粒体作为IRI中最早也是最容易受到损伤的细胞器,同时也是细胞坏死和细胞调亡发生的中心环节。通过实验研究IPo是否可以保护线粒体的正常形态、功能及其可能的机制研究,研究内容包括：透射电镜观察大鼠小肠粘膜细胞线粒体超微形态学改变和胞内总体ROS、线粒体膜电势、线粒体质量(Mitochondrial mass)、小肠粘膜细胞氧消耗等线粒体功能指标以及进行MtDNA拷贝数及损伤率的检测。各实验数据采用对比研究方法进行统计处理。结果：第二章：IPo能减轻小肠IRI后的细菌移位,肠外组织的细菌菌落数明显低于IR组(P<0.05)；而且IPo组小肠病理损伤程度也明显低于IR组(P<0.05)。第三章：IPo组与IR组比较,IPo能有效抑制小肠粘膜细胞线粒体的病理损伤,对线粒体的功能具有一定的抗损伤作用,我们的实验研究具体表现在以下几个方面：1、IPo可减轻大鼠小肠IRI模型小肠粘膜细胞线粒体超微结构的损伤；2、IPo能减轻线粒体膜蛋白的氧化损伤、提高线粒体呼吸链复合物的活性,产生对小肠的保护作用；3、IPo能提高小肠IRI后ATP的合成能力；4、IPo能保护呼吸链复合物Ⅰ-Ⅳ的活性,改善线粒体呼吸功能；5、IPo能减轻线粒体膜的脂质过氧化,保护线粒体内膜的完整性,从而维持线粒体膜电势(△Ψm)的稳定性；6、IPo能减少线粒体内源性ROS的生成,减轻氧化应激反应,产生对小肠的保护作用；7、IPo能减轻由于IRI导致的小肠粘膜细胞内线粒体蛋白的表达降低,维持线粒体质量稳定；8、IPo能减轻MtDNA的氧化损伤、减少MtDNA损伤率,同时增加MtDNA拷贝数,产生对小肠的保护作用。在本研究中,IPo和IPr两种方法的保护作用相当,两者在以上各个方面与IR组比较均具有统计学差异(P<0.05),IPo组及IPr组间比较没有显著性差异(P>0.05)。可能的机理是两者均能对再灌注小肠粘膜细胞ROS及脂质过氧化反应产物起到抑制作用,两者均能保护小肠粘膜细胞对抗IRI。由于IPo更能在临床中应用,因此认为IPo对抗组织或器官IRI的研究及应用更具有重要的现实意义。结论：1.IPo能有效减轻小肠IRI后的细菌移位、保护小肠粘膜的病理损伤以及减轻炎症因子释放；2.IPo能从多个方而减轻小肠IRI的粘膜细胞线粒体损伤程度,从而有效保护小肠粘膜线粒体的形态和功能,减少炎症因子及氧自由基的释放,抑制IRI对组织细胞的细胞调亡和坏死。更多还原

【Abstract】 Background and objective Ischemia-reperfusion injury (IRI) of the intestine is an important factor associated with a high morbidity and mortality in both surgical and trauma patients. It is of importance in situations such as the interruption of blood flow to the gut as in abdominal aortic aneurysm surgery, cardiopulmonary bypass, strangulated hernias, necrotizing enterocolitis, and intestinal transplantation. IRI of the intestine also occurs in septic and hypovolemic shock. Interruptionof blood supply results in ischemic injury which rapidly damages metabolically active tissues. Paradoxically, restoration of blood flow to the ischemic tissue initiates a cascade of events that may lead to additional cell injury known as reperfusion injury. This reperfusion damage frequently exceeds the original ischemic insult. On restoration of the blood supply, the molecular and biochemical changes that occur during ischemia predispose to free radical-mediated damage. The reduction of blood supply results in damage to the intestinal mucosa.Among the internal organs, the intestine is probably the most sensitive to IRI. The intestine is composed of labile cells that are easily injured by episodes of ischemia. Subsequent reperfusion of the intestine results in further damage to the mucosa. It has been shown that the enterocytes that are located at the tips of the villi are more sensitive to the effect of ischemia. Earlier studies attributed this increased sensitivity of the enterocyte at the tips of the villi to the location which is at the end of the distribution of a central arteriole which can lead to lower oxygen tension compared to other cells. There is substantial evidence that the mucosa of the intestine becomes the site for the production of various acute-phase proteins and cytokines. These not only influence the intestine but also may affect the function and integrity of distant organs. The initial site of abnormality in ischemia has been emphasized on the cellular mitochondria, which is particularly important in producing adenosine triphosphate (ATP) for organ recovery (16). The damage, however, is dramatically magnified by a large number of events, such as oxygen free radical formation, release of iron storage, damage of the microvasculature of IR organs, inflammatory cytokines, complement activation, and neutrophil infiltration at the site of injury. So the I/RI was regarded as one of the "motor of MODS" in the systemic inflammatory response syndrome (SIRS) and multi-organ dysfunction syndrome(MODS).The Ischemic Preconditioning (IPC) refers to a phenomenon whereby exposure of a tissue to brief periods of ischemia protects them from the deleterious effects of prolonged IRI. Its beneficial effects have been demonstrated in the liver, skeletal muscle, brain, heart, lung, intestine, and kidney in various animal models. Although the patients always have ischemic injury before in-hospital, they can not be dealed with the IPr. So the IPr has its limited in clinical practice. For this reason the ischemic postconditioning (IPo) was induced and several experiments demonstred that the IPo can relieve the injury of the IRI and it seemed good perspective of clinical application. However, the mechanism of the IPo is unknown.This study was to establish the model of ischemia-reperfusion of intestine. The aim is to study the protective effect of the ischemic postconditioning on bacteria translocation, intestinal pathology, and morphology and function of mitochondria. We hope find a good kind of approach and experimental proof to treat the intestinal ischemia-reperfusion injury.Methods The Sprague-Dawley rats were randomly divided into different groups to compare" study. All the animals divided into sham group (S), ischemia-reperfusion group (IR), ischemic preconditioning group (IPr) and ischemic postconditioning group (IPo). The S group as for sham-operated. The IR group for the rats submitted to45-minute of intestinal ischemia and2-hour or4-hour reperfusion. The IPr group was given three alternative cycles of2-min ischemia and2-min reperfusion before the intestinal ischemia. The other operation just like as the IR. group. The IPo group was given three alternative cycles of2-min reperfusion and2-min ischemia before the reperfusion. The other operation like as the IR group. The second chapter to study the protective effect of the IPo on the IRI in rats. The exam of this part include bacterial translocation, histopathologic grades, inflammatory cytokines and cells apoptosis and so on. The third chapter was to study the effect of the IPo on the mitochondria. The mitochondrion is the most sensitive cell organelle to the IRI and is the center priming place of cell apoptosis and cell necorosis. This experiment was showed that the IPo can effectively prevent the function of the mitochondria from IRI in small intestine. All of the experiment data were compared by statistics method.Result The second chapter showed that the Ecoli DH5a at lymphonodi mesenterici. liver, lung, spleen and portal vein translocation in the IPo group was lower than in the IR group at2-hour and4-hour(.P<0.05). And the intestinal pathologic injury by Chiu"s classification in the IPo group were2.30±0.96at2-hour,3.1±0.66at4-hour, which were lower than the IR group3.50±0.85at2-hour,4.62±1.14at4-hour. They have conspicuous statistic difference when compared respectively at the same time(.P<0.05). The third chapter showed that the IPo can lessen pathologic injury of mitochondria and suppress release of inflammatory factor, protect the mitochondrial function and prevent MtDNA disassociation. The contents of study in this chapter include follow aspect.1.The IPo can relieve the injury degree of mitochondria ultrastructure in cells of mucous membrane of small intestine in model of the intestinal IRI.2. The IPo can lessen oxidative damage of mitochondria membrane and elevate the compound activities of mitochondrial respiratory chain.3. The IPo can increase the synthetical abilities of adenosine triphosphate after intestinal IRI.4. The IPo can protect the activities of respiratory compound I-IV and improve mitochondrial respiratory function.5. The IPo can maintain the stability of mitochondrial membrane potential(Δ Ψm) through lighten the mitochondrial lipid peroxidation and safeguard integrality of mitochondrial inner membrane.6. The IPo can reduce generation of mitochondrial endogenous reactive oxygen species(ROS) so relieve reaction of oxidative stress.7. The IPo can maintain mitochondrial protein expression and keep mitochondrial quality while intestinal IRI.8. The IPo can relieve oxidative damage of mitochondrial DNA(mtDNA), reduce injury rate of mtDNA and increase mtDNA’s copy number. The upper aspects have significant difference in IPo and IR group (P<0.05). The IPo and IPr groups have not significant difference(P>0.05).Conclusion1. The IPo can effectively relieve the bacterial translocation and intestinal mucous membrane pathological injury, and can restrain release of pro-inflammatory factors in IRI.2. The IPo can maintain the mitochondrial form and function in IRI through restrain generation of reactive oxygen species and prevent cellular apoptosis and necrosis in IRI.更多还原