缺血后处理对大鼠小肠缺血—再灌注损伤治疗的实验机制研究

发布时间：2018-01-04 03:28

本文关键词：缺血后处理对大鼠小肠缺血—再灌注损伤治疗的实验机制研究　出处：《昆明医科大学》2012年博士论文　论文类型：学位论文

【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(P0.05)；而且IPo组小肠病理损伤程度也明显低于IR组(P0.05)。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(P0.05),IPo组及IPr组间比较没有显著性差异(P0.05)。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。
[Abstract]:Background and objective: intestinal ischemia reperfusion injury (Ischemia Reperfusion, Injury, IRI) is an important factor in morbidity and mortality of surgical and trauma patients. For example in abdominal aortic aneurysm, cardiopulmonary bypass, strangulated hernia, neonatal enteritis and intestinal necrosis of the small intestine transplantation blood flow interruption, IRI is an important stage of very common, in septic and hypovolemic shock, intestinal I RI often occur. Interrupt the blood supply to the ischemic injury caused by local effects easy to metabolically active tissue, paradoxically, will launch a series of events to restore blood flow to ischemic tissue, resulting in further cell damage, i.e. reperfusion injury. The severity of reperfusion injury are often more than the original. Ischemic injury prone to free radical mediated damage in IRI, molecular and biochemical changes. Because of this, blood supply The reduction or interruption and the subsequent recovery of blood supply will cause serious damage to the tissues and organs, namely, ischemia reperfusion injury (IRI)
In the internal organs, the small intestine is one of the most sensitive organs in IRI. Ischemia is easy to damage by unstable cells composed of intestinal tract, subsequent blood reperfusion would further damage of intestinal mucosa cells. Studies have shown that, the increase of sensitivity in the top of intestinal ischemia of intestinal epithelial cells is due to the central artery at the end, with low oxygen pressure, is sensitive to ischemia. At the same time, the main place of the small intestine is also produce a variety of acute phase proteins and inflammatory cytokines in small intestine, IRI will not only affect the small intestine itself, affect the function and integrity of remote organs can affect cell mitochondrial abnormalities. In the starting area is mainly due to the ischemia. Especially the effects of adenosine triphosphate (Adenosine Triphosphate, ATP) which causes the cells to change the hypoxic pathology, with further aggravate ischemia and hypoxia and the subsequent After reperfusion injury, the small intestine will produce a lot of harmful substances such as oxygen free radicals, damage microvessels, release inflammatory cytokines, activate complement and activate neutrophils. Therefore, intestinal IRI is considered to be one of the main dynamic factors of SIRS or MODS.
Studies have shown that ischemic preconditioning (Ischemic Preconditioning, IPr) in organ ischemia prior to one or more brief episodes of ischemia and reperfusion treatment can relieve the heart, brain, intestine, kidney and other organ IRI, but because IRI is common in small bowel volvulus, superior mesenteric artery thrombosis formation, shock, organ transplantation and heart failure, patients in occurrence of intestinal ischemia after treatment, it is difficult to pretreatment, which greatly limits its clinical application. Therefore, there is research implementation in ischemia ischemic postprocessing (Ischemic Postconditioning. IPo), which is at the beginning of reperfusion after ischemia was given once or repeatedly transient ischemia reperfusion cycles of treatment, can reduce reperfusion injury, which has better clinical prospects. But the newly discovered endogenous protective mechanism has not yet fully understood.
The aim of this study is to establish a rat IRI model of small intestine, and to study the effects of IPo on bacterial translocation, intestinal pathological changes, intestinal mucosal mitochondria morphology and function, in order to provide some theoretical and experimental basis for the clinical treatment of small intestinal IRI, and further study the protective mechanism of IPo.
Methods: the experiment was selected to establish intestinal IRI model, SPF SD rats with IPr, IPo and blank control group and IR group were compared. The second chapter studies IPo on intestinal bacterial translocation and the protective effect of pathological injury: give mark before the experiment conditions for pathogens by gavage, through experiments for different groups of intestine international organizations on the intestinal bacterial translocation detection, and pathological grading to understand the protective effect of IPo on intestinal mucosal mechanical barrier injury. The third chapter studied the protective effect of IPo on intestinal mucosal mitochondrial IRI rats: mitochondrial IRI as the earliest and most vulnerable to cell injury, at the same time cell necrosis and apoptosis. The key of the occurrence through the normal morphology experimental study whether IPo can protect the mitochondrial function, and its mechanism may be the contents of the study include: electric transmission Microscopic observation of rat intestinal mucosal cell ultrastructure changes of mitochondria and intracellular total ROS, mitochondrial membrane potential and mitochondrial mass (Mitochondrial mass), mitochondrial function index of oxygen consumption and mucosal cells in the small intestine of MtDNA copy number and the damage rate of detection. The experimental data conducted by comparing statistical processing.
Results: the second chapter: IPo can reduce the intestinal bacterial translocation after IRI, the number of bacteria in the intestinal tissue was significantly lower than that of IR group and IPo group (P0.05); small intestine pathological injury degree is significantly lower than that in IR group (P0.05). Chapter third: IPo group compared with IR group, the pathological damage of IPo can effectively inhibit the mitochondrial small intestine mucous membrane cells, has anti injury effect on mitochondrial function, specific performance of our experimental study in the following aspects: 1, IPo can reduce the rat intestinal IRI model mitochondrial ultrastructure of intestinal mucosa injury; 2, IPo can reduce oxidative damage to mitochondrial membrane protein, improve the mitochondrial respiratory chain complex activity, have a protective effect on the small intestine; 3, IPo can improve the synthesis ability of intestinal IRI ATP; 4, IPo can protect the respiratory chain complex I IV activity, improve mitochondrial respiratory function; 5, IPo can reduce the mitochondrial membrane The lipid peroxidation, protecting the integrity of the inner mitochondrial membrane, thereby maintaining the mitochondrial membrane potential (lpli m) stability; 6, IPo can reduce the generation of endogenous mitochondrial ROS, reduce oxidative stress, have a protective effect on the small intestine; 7, IPo can reduce the expression of mitochondrial protein decreased due to intestinal mucosa caused by IRI cells in the maintenance of mitochondrial quality stability; 8, IPo can reduce the MtDNA oxidative damage, reduce the damage rate of MtDNA, while increasing the copy number of MtDNA, have a protective effect on the intestine. In this study, IPo and IPr two kinds of methods of protecting function, both in each IR group compared with statistical difference (P0.05), there was no significant difference between IPo group and IPr group (P0.05). The possible mechanism is both to inhibit reperfusion intestinal mucosal cell ROS and lipid peroxidation product, both of them can. Intestinal mucosal cells against IRI. can be used in clinic because of IPo. Therefore, it is more important for IPo to study and apply IRI against tissues or organs.
Conclusion: 1.IPo can effectively reduce the intestinal bacterial translocation after IRI, protect the intestinal mucosa pathological damage and reduce the release of inflammatory factors; the degree of mucosal mitochondrial 2.IPo damage from multiple parties and reduce the intestinal IRI, so as to effectively protect the morphology and function of intestinal mucosal mitochondria, reduce inflammatory factor and oxygen free radical release inhibition IRI on the cell apoptosis and necrosis.

【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R656.7;R-332

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