本文選題：一氧化碳 + 膿毒癥　； 參考：《東南大學》2016年博士論文

【摘要】：背景和目的:膿毒癥是由感染或者可疑感染引起的全身性炎癥反應綜合征,常伴有氧化損傷、凝血功能障礙、組織灌注不足和嚴重的腸屏障功能障礙。其是創(chuàng)傷外科和ICU常見且嚴重的臨床綜合征,并可進一步發(fā)展為嚴重膿毒癥、膿毒癥性休克以及多器官功能障礙綜合征(Multiple OrganDysfunction Syndrome,MODS),被認為是重癥監(jiān)護室(Intensive Care Unit,ICU)患者的首位死亡原因。隨著人們對疾病的認識和治療措施的改善,膿毒癥的死亡率開始下降,但嚴重膿毒癥和膿毒癥性休克的死亡率仍然較高。因此,進一步尋找對嚴重膿毒癥及其并發(fā)的MODS新的治療措施,是目前臨床上迫切需要解決的問題。腸道是膿毒癥損傷受累最早的器官之一,也是應激中心,腸道受累導致腸黏膜屏障功能障礙,進而導致持續(xù)性的腸源性細菌和毒素進入組織和血液,誘導全身過度失控炎癥反應,參與遠隔器官損傷及MODS的發(fā)生和發(fā)展。已有研究證實,維持緊密連接的完整性和腸黏膜上皮細胞的正常功能已成為治療膿毒癥和MODS的關鍵所在。近來研究證實,一氧化碳(Carbon Monoxide,CO)作為一種細胞信使分子在生物體內(nèi)具有抗炎、抗凋亡、舒張血管和抑制血小板聚集等多種生理學作用。在動物實驗中,增加組織CO的含量能改善各種各樣的胃腸道疾病,如糖尿病性胃輕癱、炎癥性腸病(Inflammatory Bowel Disease,IBD)、術后腸梗阻以及小腸移植。本研究通過建立盲腸結(jié)扎穿孔(Cecal Ligation And Puncture,CLP)大鼠膿毒癥模型,給予外源性CO釋放分子2(Carbon Monoxide-Releasing Molecule-2,CORM-2),觀察膿毒癥大鼠細胞因子形成、腸黏膜超微結(jié)構改變、緊密連接蛋白表達等的影響,并從細胞和分子水平探討其可能的作用機制,為臨床防治膿毒癥的發(fā)生發(fā)展提供新的參考。本研究分兩個部分:第一部分,外源性一氧化碳對膿毒癥大鼠腸黏膜屏障損傷的保護作用;第二部分,外源性一氧化碳對膿毒癥大鼠腸黏膜屏障損傷保護作用的機制研究。方法:(1).SD大鼠隨機分為4組:假手術組(Sham);CLP組;CLP大鼠加CORM-2治療組(CORM);CLP大鼠加無活性CORM-2(iCORM-2)治療組(iCORM)。CLP24小時后收集血液和小腸組織標本。ELISA檢測血清中TNF-α和IL-1β含量;光鏡觀察小腸組織病理形態(tài)變化;電鏡觀察小腸組織超微結(jié)構變化;Western blot檢測小腸黏膜緊密連接蛋白(ZO-1,Claudin-1和Occludin)表達;免疫熒光觀察緊密連接蛋白分布情況;FITC熒光標記法觀察小腸通透性。并比較觀察實驗各組72小時生存率。(2).檢測血清二胺氧化酶(DAO)活性、D-乳酸含量和小腸黏膜髓過氧化物酶(MPO)活性,RT-PCR檢測小腸黏膜Rho激酶(ROCK)-1和ROCK-2mRNA表達,Western blot檢測小腸黏膜磷酸化的肌球蛋白磷酸酶目標亞基1(p-MYPT-1)/肌球蛋白磷酸酶目標亞基1(MYPT-1)和磷酸化的肌球蛋白輕鏈(p-MLC)/肌球蛋白輕鏈(MLC)蛋白的表達。結(jié)果:(1).相對于Sham組,CLP組、CORM組和iCORM組三組大鼠血清TNF-α和IL-1β含量明顯增加(P0.01);小腸病理損傷和緊密連接結(jié)構破壞顯著加重,Chiu's評分增加(P0.05);腸黏膜緊密連接蛋白(ZO-1,Claudin-1和Occludin)含量下降;共聚焦顯微鏡觀察腸黏膜膜緊密連接蛋白(ZO-1,Claudin-1和Occludin)分布減少;腸黏膜通透性增加(P0.05);生存率下降(P0.05)。而CORM組與CLP組、iCORM組比較,血清TNF-α和IL-1β含量降低(P0.01);小腸病理損傷和緊密連接結(jié)構破壞顯著減輕,Chiu's評分降低(P0.05);腸黏膜緊密連接蛋白(ZO-1,Claudin-1和Occludin)含量增加;共聚焦顯微鏡觀察腸黏膜膜緊密連接蛋白(ZO-1,Claudin-1和Occludin)分布增加;腸黏膜通透性降低(P0.05);生存率改善(P0.05)。(2).相對于Sham組,CLP組、CORM組和iCORM組三組大鼠血清DAO活性、D乳酸含量和腸黏膜MPO活性明顯增加(P0.01);RT-PCR顯示小腸黏膜ROCK-1和ROCK-2mRNA顯著增加(P0.05);腸黏膜MYPT-1和MLC磷酸化水平明顯增加(P0.05)。而CORM組與CLP組、iCORM組比較,血清DAO活性、D乳酸含量和腸黏膜MPO活性明顯下降(P0.01);RT-PCR顯示小腸黏膜ROCK-1顯著下降(P0.05),ROCK-2 mRNA無明顯變化;腸黏膜MYPT-1和MLC磷酸化水平明顯下降(P0.05)。結(jié)論:(1)CORM-2可以緩解CLP誘導的膿毒癥大鼠的腸道損傷,抑制炎癥介質(zhì)的釋放,保護腸上皮細胞間緊密連接,改善腸黏膜的通透性,起到保護作用。最終顯著提高大鼠的生存率。(2)CORM-2對膿毒癥大鼠腸黏膜的保護作用可能與其抑制Rho/ROCK信號通路減少相關炎性基因和蛋白表達有關。
[Abstract]:Background and objective: sepsis is a systemic inflammatory response syndrome caused by infection or suspected infection, often accompanied by oxidative damage, coagulation dysfunction, insufficiency of tissue perfusion, and severe intestinal barrier dysfunction. It is a common and severe clinical syndrome in trauma surgery and ICU, and can be further developed into severe sepsis and sepsis. Shock and multiple organ dysfunction syndrome (Multiple OrganDysfunction Syndrome, MODS) are considered the leading causes of death in the Intensive Care Unit (ICU) patients. The death rate of sepsis begins to decline with the improvement of understanding and treatment of the disease, but the death of severe sepsis and septic shock. The death rate is still high. Therefore, further finding new treatment measures for severe sepsis and its concurrent MODS is an urgent problem to be solved at present. Intestinal is one of the earliest organs involved in sepsis injury, and also a stress center. Intestinal involvement leads to intestinal mucosal barrier dysfunction, which leads to persistent intestinal bacteria. And toxins enter tissue and blood, induce Overcontrol of systemic inflammatory response, participate in distant organ damage and the occurrence and development of MODS. Studies have shown that maintaining the integrity of close connections and normal function of intestinal epithelial cells has become the key to the treatment of sepsis and MODS. Recent studies have confirmed that carbon monoxide (Carbon Monoxid) E, CO), as a cell messenger, has many physiological functions, such as anti-inflammatory, anti apoptosis, vasodilatation, and inhibition of platelet aggregation in the organism. In animal experiments, increasing the content of tissue CO can improve a variety of gastrointestinal diseases, such as diabetic gastroparesis, inflammatory bowel disease (Inflammatory Bowel Disease, IBD), and postoperative intestinal tract (IBD). Obstruction and small bowel transplantation. In this study, a rat sepsis model of Cecal Ligation And Puncture (CLP) was established, and exogenous CO release molecule 2 (Carbon Monoxide-Releasing Molecule-2, CORM-2) was given. The effects of cytokine formation, ultrastructural changes of intestinal mucosa and the expression of tight connexin in the septic rats were observed. This study provides a new reference for the development of clinical prevention and treatment of sepsis. This study is divided into two parts: the first part, the protective effect of exogenous carbon monoxide on intestinal mucosal barrier damage in septic rats; the second part, exogenous carbon monoxide on the intestinal mucosal barrier of sepsis rats Methods: (1).SD rats were randomly divided into 4 groups: sham operation group (Sham), group CLP, CLP rats and CORM-2 treatment group (CORM); CLP rats and non active CORM-2 (iCORM-2) treatment group (iCORM) were used to collect blood and small intestine tissue specimens. Pathological changes; ultrastructural changes of small intestinal tissue were observed by electron microscopy; Western blot was used to detect the expression of compact connexin (ZO-1, Claudin-1 and Occludin) in small intestinal mucosa; the distribution of tight connexin was observed by immunofluorescence; the small intestinal permeability was observed by FITC fluorescence labeling, and the 72 hour survival rate was compared with that of the observed groups. (2). The detection of serum two Amines oxidase (DAO) activity, D- lactic acid content and myeloperoxidase (MPO) activity of small intestinal mucosa, RT-PCR detection of Rho kinase (ROCK) -1 and ROCK-2mRNA expression in small intestinal mucosa, Western blot detection of myosin phosphatase target subunit 1 (p-MYPT-1) / myosin phosphatase target subunit 1 (MYPT-1) and phosphorylated myocutaneous eggs The expression of white light chain (p-MLC) / myosin light chain (MLC) protein. Results: (1) compared to group Sham, the content of TNF- alpha and IL-1 beta in serum of three groups of group CLP, CORM and iCORM increased significantly (P0.01); the pathological damage and close connection of the small intestine increased significantly, the Chiu's score increased (P0.05); the intestinal mucosa tightly connexin Cludin) decreased; confocal microscopy showed a decrease in the distribution of close connexin (ZO-1, Claudin-1 and Occludin) in the intestinal mucosa; intestinal mucosal permeability increased (P0.05); the survival rate decreased (P0.05). The CORM group was compared with the CLP group, the iCORM group, and the serum TNF- A and IL-1 beta content decreased (P0.01); the damage of the small intestine and the destruction of the close connexion structure were significantly reduced. Light, Chiu's score decreased (P0.05), intestinal mucosal tight connexin (ZO-1, Claudin-1 and Occludin) increased; confocal microscopy was used to observe the distribution of tight connexin (ZO-1, Claudin-1 and Occludin) in intestinal mucosa; intestinal mucosal permeability (P0.05) and survival rate (P0.05). (P0.05). (2). Three groups of CLP, CLP, and groups Serum DAO activity, D lactic acid content and intestinal mucosa MPO activity increased significantly (P0.01), RT-PCR showed that ROCK-1 and ROCK-2mRNA in intestinal mucosa increased significantly (P0.05), MYPT-1 and MLC phosphorylation in intestinal mucosa increased significantly (P0.05). RT-PCR showed significant decrease in ROCK-1 of small intestinal mucosa (P0.05), no significant changes in ROCK-2 mRNA, and significantly decreased phosphorylation of MYPT-1 and MLC in intestinal mucosa (P0.05). Conclusion: (1) CORM-2 can alleviate intestinal damage in rats induced by CLP, inhibit the release of inflammatory mediators, protect the close connections between intestinal epithelial cells, and improve intestinal permeability, The protective effect of CORM-2 on the intestinal mucosa of rats with sepsis may be associated with the inhibition of Rho/ROCK signaling pathway related inflammatory genes and protein expression.
【學位授予單位】：東南大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R459.7

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