【摘要】：腸缺血再灌注（ischemia/reperfusion, I/R）引起的腸系膜上動脈閉塞性（superior mesenteric artery occlusion, SMAO）休克，是臨床常見的危重病理過程，常見于休克復(fù)蘇、器官移植術(shù)后、嚴重創(chuàng)傷救治過程等。腸I/R引起腸道屏障功能障礙、細菌/內(nèi)毒素移位和大量炎癥介質(zhì)釋放，誘發(fā)多器官功能障礙綜合征（multiple organ dysfunctionsyndrome, MODS），甚至危及患者生命。因此，探討腸道I/R引起遠隔器官損傷的機制以及尋找有效的干預(yù)措施，值得深入研究。 隨著對腸淋巴途徑研究的深入，越來越多的結(jié)果表明，腸淋巴途徑在腸I/R導(dǎo)致遠隔器官損傷的發(fā)病學(xué)中具有重要作用。我所前期研究結(jié)果表明，腸淋巴再灌注（mesenteric lymph reperfusion, MLR）可加重SMAO休克大鼠肺、腎、心、肝等器官的組織學(xué)損傷，其作用機制與加劇自由基損傷、炎癥損傷有關(guān)。為了進一步探討MLR加重SMAO休克大鼠炎癥反應(yīng)的作用機制，本研究以中性粒細胞（polymerphonuclear neutrophil, PMN）、高遷移率族蛋白-1（highmobility group protein box-1, HMGB1）、晚期糖基化產(chǎn)物受體（receptorof advanced glycation end-products, RAGE）、核因子-κB（nuclearfactor-kappa B, NF-κB）為切入點，探討啟動炎癥反應(yīng)的多種因素在MLR加重SMAO休克器官損傷中的作用機制，為SMAO休克器官損傷的防治提供實驗依據(jù)。 24只Wistar雄性大鼠，隨機分為4組：SMAO組，夾閉腸系膜上動脈（superior mesenteric artery, SMA）1h，再灌注2h；MLR組，，夾閉腸系膜淋巴管（mesenteric lymphatic duct, MLD）1h，再灌注2h;SMAO+MLR組，同時夾閉MLD和SMA 1h，再灌注2h；假手術(shù)組（Sham組），在SMA與MLR下穿線。于再灌注2h后，選擇固定位置留取心、肝、肺、腎組織，一部分用中性甲醛固定，石蠟包埋，切片后應(yīng)用免疫組織化學(xué)染色方法觀察各組織HMGB1、RAGE、NF-κBp65和髓過氧化物酶（myeloperoxidase, MPO）表達水平；另外一部分組織制備16.7%組織勻漿，應(yīng)用酶聯(lián)免疫方法檢測RAGE、細胞間黏附分子-1（intercellular adhesion molecule-1, ICAM-1）含量。 實驗結(jié)果顯示，MLR和sham組的各項指標未見統(tǒng)計學(xué)差異。SMAO組肺、腎、心、肝組織的ICAM-1含量顯著高于MLR和sham組(P0.01)、MPO表達水平強于MLR和sham組(P0.05, P0.01)，SMAO+MLR組肺、腎、心、肝組織的ICAM-1含量顯著高于SMAO、MLR和sham組(P0.01)、MPO表達水平在不同程度上強于SMAO、MLR和sham組(P0.05, P0.01)，結(jié)果提示MLR加重SMAO休克大鼠器官炎癥反應(yīng)的機制與PMN扣押于組織增多有關(guān)。SMAO組、SMAO+MLR組肺、腎、心、肝組織RAGE的含量以及HMGB1、RAGE表達均高于或強于MLR和Sham組(P0.05, P0.01)，且SMAO+MLR組的這些指標高于或強于SMAO組(P0.05, P0.01)，結(jié)果提示MLR加重SMAO休克大鼠器官炎癥反應(yīng)的機制與HMGB1表達增強有關(guān)。同時也發(fā)現(xiàn)，SMAO組、SMAO+MLR組肺、腎、心、肝組織的NF-κB表達均顯著強于MLR和sham組(P0.05, P0.01)，且SMAO+MLR組各組織的NF-κB表達強于SMAO組(P0.05, P0.01)，結(jié)果表明，MLR加重SMAO休克大鼠器官炎癥反應(yīng)的機制與NF-κB表達增強有關(guān)。 綜上，一方面，MLR增加了SMAO休克大鼠各組織器官的ICAM-1表達、引起PMN黏附、扣押于組織中，過多的PMN活化加重了機體炎癥反應(yīng)；另一方面，MLR增加了SMAO休克大鼠各組織器官的HMGB1表達、RAGE含量與表達、NF-κB表達，從而加重了由HMGB1、RAGE、NF-κB導(dǎo)致的炎癥反應(yīng)；至于HMGB1、RAGE、NF-κB三者之間的關(guān)系，以及在MLR加重SMAO休克大鼠炎癥反應(yīng)中的相互作用，還需要在以后的研究中進一步驗證。
[Abstract]:Superior mesenteric artery occlusion (SMAO) shock caused by ischemia/reperfusion (I/R) is a common critical pathological process in the clinic. It is common in shock resuscitation, organ transplantation, and severe trauma treatment. Intestinal I/R causes intestinal barrier dysfunction and bacterial / endotoxin translocation. The release of a large number of inflammatory mediators can induce multiple organ dysfunctionsyndrome (MODS) and even endanger the patient's life. Therefore, it is worthy of further study to explore the mechanism of intestinal I/R caused by the injury of distant organs and to find effective intervention measures.
With the further study of the intestinal lymphatic pathway, more and more results show that the intestinal lymphatic pathway plays an important role in the pathogenesis of I/R induced distant organ damage. The results of my previous study showed that intestinal lymphatic reperfusion (mesenteric lymph reperfusion, MLR) could aggravate the histological damage of lung, kidney, heart and liver in SMAO shock rats. Injury, its mechanism is related to the aggravation of free radical damage and inflammatory injury. To further explore the mechanism of MLR aggravating the inflammatory reaction in SMAO shock rats, this study uses neutrophils (polymerphonuclear neutrophil, PMN), high mobility group protein -1 (HighMobility group protein box-1, HMGB1), and late glycosylated products receptor (recept) Orof advanced glycation end-products, RAGE), nuclear factor kappa B (nuclearfactor-kappa B, NF- kappa B) as the cut in point, to explore the mechanism of initiating inflammatory reaction in MLR aggravated SMAO shock organ damage, providing experimental basis for the prevention and treatment of shock organ damage.
24 male Wistar rats were randomly divided into 4 groups: group SMAO, superior mesenteric artery, SMA 1H, and 2H, MLR group, and then the mesenteric lymphatic vessels (mesenteric lymphatic duct), then reperfusion; After reperfusion of 2h, a fixed position was selected to leave the heart, liver, lung, and kidney tissue, part of which was fixed with neutral formaldehyde and embedded in paraffin. After the section, the expression of HMGB1, RAGE, NF- kappa Bp65 and myeloperoxidase (MPO) was observed by immunohistochemical staining. The other part of the tissue was used to prepare 16.7% tissue homogenates. Enzyme linked immunosorbent assay (ELISA) was used to detect the content of RAGE and intercellular adhesion molecule-1 (ICAM-1) in -1.
The experimental results showed that the indexes of MLR and sham group were not statistically different in group.SMAO, and the content of ICAM-1 in kidney, heart and liver tissue was significantly higher than that in group MLR and sham (P0.01). The expression level of MPO was stronger than that of MLR and sham groups (P0.05, P0.01). SMAO, MLR and sham group (P0.05, P0.01). The results suggest that MLR aggravates the mechanism of organ inflammation in SMAO shock rats and PMN seizures in group.SMAO, SMAO+MLR group lung, kidney, heart, liver tissue RAGE content and HMGB1. Higher or stronger than group SMAO (P0.05, P0.01), the results suggested that the mechanism of MLR aggravated the organ inflammatory response of SMAO shock rats was related to the enhancement of HMGB1 expression. Meanwhile, the expression of NF- kappa B in the lung, kidney, heart and liver tissues of group SMAO was significantly stronger than that in MLR and sham groups. .05 (P0.01), the results showed that MLR aggravated organ inflammatory response in SMAO shock rats and increased expression of NF- kappa B.
On the one hand, MLR increased the expression of ICAM-1 in the tissues and organs of SMAO shock rats, resulting in PMN adhesion and seizure in the tissue, and excessive PMN activation aggravated the inflammatory response of the body; on the other hand, MLR increased the HMGB1 expression in the tissues and organs of the SMAO shock rats, and the RAGE content and expression, NF- kappa B expression, thus aggravated HMGB1, HMGB1, HMGB1, kappa concerned. The associated inflammatory response; the relationship between HMGB1, RAGE, NF- kappa B three and the interaction of MLR in the inflammatory response to SMAO shock rats need to be further verified in future studies.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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