本文關(guān)鍵詞：缺血后處理對(duì)大鼠小腸缺血—再灌注損傷治療的實(shí)驗(yàn)機(jī)制研究　出處：《昆明醫(yī)科大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 缺血再灌注損傷 小腸 缺血后處理 缺血預(yù)處理 大鼠

【摘要】：背景及目的：小腸缺血再灌注損傷(Ischemia Reperfusion Injury, IRI)是外科手術(shù)和創(chuàng)傷病人高發(fā)病率和死亡率的重要因素。例如在腹主動(dòng)脈瘤、心肺旁路手術(shù)、絞窄性疝、新生兒壞死性小腸炎和小腸移植等腸血流量中斷的情況下,IRI是非常常見的重要階段,在敗血癥和低血容量性休克時(shí),小腸I RI也經(jīng)常發(fā)生。血流供應(yīng)的中斷造成局部的缺血損傷易于影響代謝活躍的組織,相矛盾的是,缺血組織血流恢復(fù)時(shí)將會(huì)啟動(dòng)一系列的事件,導(dǎo)致細(xì)胞進(jìn)一步的損傷,即再灌注損傷。再灌注損傷的嚴(yán)重性往往超過了最初的缺血性損傷。在IRI中易于發(fā)生自由基介導(dǎo)的損傷,發(fā)生分子和生化等變化。因此,血液供應(yīng)減少或中斷以及隨后的血供恢復(fù)將造成組織器官的嚴(yán)重?fù)p害,即缺血再灌注損傷(IRI) 在內(nèi)臟器官中,小腸是對(duì)IRI最敏感的器官之一。缺血時(shí)容易損傷由不穩(wěn)定細(xì)胞組成的腸道,隨后的血液再灌注則會(huì)進(jìn)一步損傷小腸粘膜細(xì)胞。有研究認(rèn)為,位于絨毛的頂端腸上皮細(xì)胞對(duì)缺血敏感性增加的原因是由于其位于中央動(dòng)脈的盡頭,具有較低氧分壓,對(duì)局部缺血較敏感。同時(shí),小腸也是產(chǎn)生各種急性期蛋白和細(xì)胞炎癥因子的主要場(chǎng)所,小腸IRI不僅會(huì)影響小腸本身,還能影響遠(yuǎn)隔器官的功能和完整性。在缺血發(fā)生起始區(qū)域的異常改變主要是影響細(xì)胞線粒體,特別是影響三磷酸腺苷(Adenosine Triphosphate, ATP)的產(chǎn)生,導(dǎo)致細(xì)胞發(fā)生缺氧性病理改變,隨著缺血缺氧的進(jìn)一步加重以及隨之而來的再灌注損傷,小腸將產(chǎn)生大量的氧自由基等有害物質(zhì),損傷微血管、釋放炎性細(xì)胞因子、激活補(bǔ)體和活化中性粒細(xì)胞。因此,小腸IRI被認(rèn)為是SIRS或MODS最主要的使動(dòng)因素之一 有研究表明缺血預(yù)處理(Ischemic Preconditioning, IPr)即在器官缺血前給予一次或多次短暫的缺血與再灌注處理,可減輕心、腦、小腸、腎等多種器官的IRI,但由于小腸IRI常見于腸扭轉(zhuǎn)、腸系膜上動(dòng)脈血栓形成、休克、器官移植術(shù)和心力衰竭等,臨床上患者多在發(fā)生腸缺血后就診,難以進(jìn)行預(yù)處理,這就極大地限制了其臨床應(yīng)用。因此,有人研究在缺血發(fā)生后實(shí)施的缺血后處理(Ischemic Postconditioning. IPo),即在缺血后再灌注開始時(shí)立刻給予一次或多次短暫再灌注與缺血交替循環(huán)處理,可以減輕再灌注損傷,具有更好的臨床應(yīng)用前景。但這一新發(fā)現(xiàn)的內(nèi)源性保護(hù)機(jī)制尚未完全闡明。 本研究通過建立大鼠小腸IRI模型,研究IPo對(duì)細(xì)菌移位、小腸病理改變、小腸粘膜線粒體形態(tài)、功能進(jìn)行研究,以期為臨床治療小腸IRI提供一定的理論和實(shí)驗(yàn)依據(jù),并對(duì)IPo的保護(hù)性機(jī)制做進(jìn)一步研究。 方法：實(shí)驗(yàn)選用SPF級(jí)SD大鼠建立小腸IRI模型,采用IPr、IPo與空白對(duì)照組和IR組進(jìn)行對(duì)比研究。第二章研究IPo對(duì)小腸細(xì)菌移位及病理損傷的保護(hù)作用：通過實(shí)驗(yàn)前給予標(biāo)記的條件致病菌灌胃,實(shí)驗(yàn)通過對(duì)不同組別的腸外組織進(jìn)行細(xì)菌移位檢測(cè),以及對(duì)小腸病理損傷分級(jí)評(píng)分來了解IPo對(duì)小腸粘膜機(jī)械屏障損傷的保護(hù)作用。第三章進(jìn)行IPo對(duì)大鼠小腸IRI粘膜細(xì)胞線粒體保護(hù)作用的實(shí)驗(yàn)研究：線粒體作為IRI中最早也是最容易受到損傷的細(xì)胞器,同時(shí)也是細(xì)胞壞死和細(xì)胞調(diào)亡發(fā)生的中心環(huán)節(jié)。通過實(shí)驗(yàn)研究IPo是否可以保護(hù)線粒體的正常形態(tài)、功能及其可能的機(jī)制研究,研究內(nèi)容包括：透射電鏡觀察大鼠小腸粘膜細(xì)胞線粒體超微形態(tài)學(xué)改變和胞內(nèi)總體ROS、線粒體膜電勢(shì)、線粒體質(zhì)量(Mitochondrial mass)、小腸粘膜細(xì)胞氧消耗等線粒體功能指標(biāo)以及進(jìn)行MtDNA拷貝數(shù)及損傷率的檢測(cè)。各實(shí)驗(yàn)數(shù)據(jù)采用對(duì)比研究方法進(jìn)行統(tǒng)計(jì)處理。 結(jié)果：第二章：IPo能減輕小腸IRI后的細(xì)菌移位,腸外組織的細(xì)菌菌落數(shù)明顯低于IR組(P0.05)；而且IPo組小腸病理損傷程度也明顯低于IR組(P0.05)。第三章：IPo組與IR組比較,IPo能有效抑制小腸粘膜細(xì)胞線粒體的病理損傷,對(duì)線粒體的功能具有一定的抗損傷作用,我們的實(shí)驗(yàn)研究具體表現(xiàn)在以下幾個(gè)方面：1、IPo可減輕大鼠小腸IRI模型小腸粘膜細(xì)胞線粒體超微結(jié)構(gòu)的損傷；2、IPo能減輕線粒體膜蛋白的氧化損傷、提高線粒體呼吸鏈復(fù)合物的活性,產(chǎn)生對(duì)小腸的保護(hù)作用；3、IPo能提高小腸IRI后ATP的合成能力；4、IPo能保護(hù)呼吸鏈復(fù)合物Ⅰ-Ⅳ的活性,改善線粒體呼吸功能；5、IPo能減輕線粒體膜的脂質(zhì)過氧化,保護(hù)線粒體內(nèi)膜的完整性,從而維持線粒體膜電勢(shì)(△Ψm)的穩(wěn)定性；6、IPo能減少線粒體內(nèi)源性ROS的生成,減輕氧化應(yīng)激反應(yīng),產(chǎn)生對(duì)小腸的保護(hù)作用；7、IPo能減輕由于IRI導(dǎo)致的小腸粘膜細(xì)胞內(nèi)線粒體蛋白的表達(dá)降低,維持線粒體質(zhì)量穩(wěn)定；8、IPo能減輕MtDNA的氧化損傷、減少M(fèi)tDNA損傷率,同時(shí)增加MtDNA拷貝數(shù),產(chǎn)生對(duì)小腸的保護(hù)作用。在本研究中,IPo和IPr兩種方法的保護(hù)作用相當(dāng),兩者在以上各個(gè)方面與IR組比較均具有統(tǒng)計(jì)學(xué)差異(P0.05),IPo組及IPr組間比較沒有顯著性差異(P0.05)�？赡艿臋C(jī)理是兩者均能對(duì)再灌注小腸粘膜細(xì)胞ROS及脂質(zhì)過氧化反應(yīng)產(chǎn)物起到抑制作用,兩者均能保護(hù)小腸粘膜細(xì)胞對(duì)抗IRI。由于IPo更能在臨床中應(yīng)用,因此認(rèn)為IPo對(duì)抗組織或器官IRI的研究及應(yīng)用更具有重要的現(xiàn)實(shí)意義。 結(jié)論：1.IPo能有效減輕小腸IRI后的細(xì)菌移位、保護(hù)小腸粘膜的病理損傷以及減輕炎癥因子釋放；2.IPo能從多個(gè)方而減輕小腸IRI的粘膜細(xì)胞線粒體損傷程度,從而有效保護(hù)小腸粘膜線粒體的形態(tài)和功能,減少炎癥因子及氧自由基的釋放,抑制IRI對(duì)組織細(xì)胞的細(xì)胞調(diào)亡和壞死。
[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.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R656.7;R-332

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