本文選題：腹腔高壓癥 + 失血性休克��； 參考：《第三軍醫(yī)大學》2016年博士論文

【摘要】：研究背景嚴重創(chuàng)傷性患者在失血性休克、燒傷、腹部創(chuàng)傷等,在救治過程中常發(fā)生腹腔高壓癥(intra-abdominal hypertension,IAH),甚至進展為腹腔間隙綜合征(abdominal compartment syndrome,ACS),累及多種腹腔內(nèi)臟器(腸道、胃、肝和腎)及腹腔外器官(腦、心及肺)等。IAH/ACS包括:原發(fā)性、繼發(fā)性和復發(fā)性3類。原發(fā)性IAH/ACS是由腹腔/盆腔疾病或損傷所引起;繼發(fā)性IAH/ACS并不由腹腔/盆腔疾病或損傷引起,如全身炎癥反應綜合征、缺血休克和大面積燒傷等;復發(fā)性IAH/ACS是指前期IAH/ACS經(jīng)手術或藥物治療緩解后再次發(fā)生。IAH發(fā)生的危險因素包括:創(chuàng)傷后大量輸液、復蘇后毛細血管滲漏、腹腔內(nèi)容物增加和腹壁順應性下降等,而其中創(chuàng)傷后大量輸液是主要原因。IAH的發(fā)病機制和病理進展過程目前尚不完全明確。目前治療IAH的方法依然集中在剖腹減壓等外科技術的應用上,但依然存在如減壓后綜合征造成的再灌注損傷等問題。由于腸道水腫等損傷是腹內(nèi)壓升高的重要因素,本研究以如何減緩腸道水腫等損傷的發(fā)生,以及如何緩解腹內(nèi)高壓影響的重要腹腔外器官—腦部的損傷為主要內(nèi)容。目前越來越受到人們關注的黑色素皮質(zhì)激素(melanocortin,MC),MC通過其不同受體在多器官中發(fā)揮調(diào)節(jié)色素沉著,能量穩(wěn)態(tài),炎癥控制和免疫調(diào)節(jié)等功能,有研究發(fā)現(xiàn)MC4受體(melanocortin 4 receptor,MC4R)激動劑和其人工合成類似物可以在失血性休克、缺血性卒中和創(chuàng)傷性顱腦損傷中發(fā)揮保護作用,并通過膽堿能抗炎通路發(fā)揮快速抗炎的作用,但具體機制尚不清楚。本課題旨在探尋MC是否可以起到緩解繼發(fā)性IAH中腸和腦功能障礙的作用,并進一步探尋其機制,以期為臨床腹腔高壓癥患者的治療提供新的策略。為了檢驗上述假設,本研究首先建立繼發(fā)性IAH大鼠模型,該模型應便于操作、可重復性高并可在一定程度上較好的模擬IAH的發(fā)病過程。其次,觀察給予RO27-3225(MC4R激動劑)后可否減輕IAH誘導的腸道損傷,該保護作用通過何種機制發(fā)揮效果。最后探討RO27-3225是否對于IAH誘發(fā)的腹腔外器官損害--腦損傷的具有保護效果,并判斷膽堿能信號通路在其中的作用。研究方法1.采用股動脈插管后放血法,誘導平均動脈壓(mean arterial pressure,MAP)分別達到:40mm Hg(維持120 min);30mm Hg(維持90min)和25mm Hg(維持60min),而后均合并IAP=20mm Hg(維持4 h)以建立大鼠繼發(fā)性IAH模型,利用干濕比法檢測腸道和腦組織含水量,利用HE染色法在光鏡下觀察大鼠腸道組織形態(tài)學和分析不同組間生存率,從中選擇一種既可造成明顯損傷又可保證生存率以供后續(xù)試驗可順利進行的大鼠休克后IAH模型進行下述觀察。2.在繼發(fā)性IAH大鼠模型(30mm Hg維持90min合并IAP=20mm Hg維持4h)中,通過觀察大鼠MAP的變化程度;采用real-time PCR法檢測腸壁全層炎癥因子(TNF-α和IL-1β)變化;使用干濕比法檢測腸道含水量變化;利用HE染色法在光鏡下觀察大鼠腸道組織形態(tài)學組織變化;利用酶聯(lián)免疫吸附方法檢測血漿內(nèi)腸脂肪酸結合蛋白變化;通過檢測異硫氰酸熒光素以反映腸道滲透性變化和使用免疫印跡法檢測影響腸道滲透性的關鍵因子ROCK1及肌球蛋白輕鏈磷酸化水平以及檢測腸道內(nèi)超氧化物歧化酶變化等指標探討IAH對腸道的損傷效果。并在此基礎上進一步觀察RO27-3225是否可以發(fā)揮保護腸道的作用。3.在繼發(fā)性IAH大鼠模型中,利用干濕比法檢測腦含水量變化;通過檢測伊文思藍外滲法反映血腦屏障滲透性、使用real-time PCR法和酶聯(lián)免疫吸附方法檢測腦內(nèi)炎癥因子(TNF-α和IL-1β)變化、采用酶聯(lián)免疫吸附方法及免疫印跡法檢測水通道蛋白4及金屬基質(zhì)蛋白酶9表達變化,以及利用免疫組化法檢測神經(jīng)膠質(zhì)細胞表達的變化等指標探討IAH對腦的損傷效果,進一步觀察RO27-3225是否可以發(fā)揮保護腦組織的作用。研究結果1.選取MAP為30mm Hg持續(xù)90 min合并IAP=20 mm Hg持續(xù)4 h這一條件可誘導大鼠腸道出現(xiàn)明顯組織學損傷,導致腸道含水量明顯上升,并可保證其生存率以確保后續(xù)試驗可以進行;2.RO27-3225可在一定程度上穩(wěn)定MAP變化,降低IAH所誘發(fā)的腸道內(nèi)炎癥因子(TNF-α和IL-1β)升高,緩解IAH導致的腸道內(nèi)ROCK 1蛋白表達和肌球蛋白輕鏈磷酸化水平下降,降低血漿內(nèi)腸脂肪酸結合蛋白水平,減輕腸水腫、腸道滲透性和腸道組織形態(tài)學等結果的異常變化,從而起到緩解IAH導致的腸道損傷的作用。而該保護作用可被其特異性阻滯劑(HS024)和煙堿型乙酰膽堿受體拮抗劑(氯異桑大明)所阻止。3.RO27-3225可減輕IAH導致的腦水腫,降低炎癥因子、水通道蛋白4及金屬基質(zhì)蛋白酶9的表達,從而發(fā)揮緩解IAH中腦損傷的作用。而該保護作用可被其特異性阻滯劑(HS024)和煙堿型乙酰膽堿受體拮抗劑(氯異桑大明)所阻止。但對IAH誘導的神經(jīng)膠質(zhì)細胞表達增多并無影響。結論1.IAH可導致腹腔內(nèi)臟器-腸道產(chǎn)生明顯水腫及炎癥反應,并出現(xiàn)明顯組織學損傷,此外IAH也可誘導腹腔外器官-腦組織產(chǎn)生明顯炎癥、水腫并導致血腦屏障完整性損害;2.選擇性MC4R激動劑(RO27-3225)可以穩(wěn)定機體血流動力學,并通過調(diào)節(jié)ROCK1表達和肌球蛋白輕鏈磷酸化水平以緩解腸道滲透性增高,并可降低炎癥反應程度,而有效緩解IAH誘導的腸道損傷。該保護作用可能是通過膽堿能通路發(fā)揮效果;3.選擇性MC4R激動劑可以通過降低金屬基質(zhì)蛋白酶9表達以緩解血腦屏障破壞,通過作用水通道蛋白4以降低腦水腫程度,并可減輕腦組織炎癥反應有效改善IAH導致的腦組織損傷,該保護作用可能是通過膽堿能通路發(fā)揮效果。但其在急性期內(nèi)對神經(jīng)膠質(zhì)細胞數(shù)量無明顯影響。
[Abstract]:The research background of severe traumatic patients in hemorrhagic shock, burns, abdominal trauma, and so on. In the course of treatment, intra-abdominal hypertension (IAH) often occurs, and even abdominal compartment syndrome (ACS), involving a variety of abdominal viscera (intestine, stomach, liver and kidney) and external organs of the abdomen (brain, .IAH/ACS includes 3 types: primary, secondary and recurrent. Primary IAH/ACS is caused by abdominal / pelvic diseases or injuries; secondary IAH/ACS is not caused by abdominal / pelvic diseases or injuries, such as systemic inflammatory response syndrome, ischemic shock and large area burn, and recurrent IAH/ACS refers to prophase IAH/ACS through surgery or medicine The risk factors for the recurrence of.IAH after the treatment of remission include: massive infusion after trauma, capillary leakage after resuscitation, increase of intraperitoneal content and decrease of abdominal wall compliance, and the pathogenesis and pathological progress of.IAH are not completely clear at present. At present, the methods for the treatment of IAH However, it is focused on the application of surgical techniques such as laparotomy, but there are still problems such as reperfusion injury caused by post decompression syndrome. Because of intestinal edema, such as intestinal edema is an important factor in intraperitoneal pressure increase, this study can reduce the occurrence of intestinal edema and other injuries, as well as the important external organs such as how to relieve the effect of intraabdominal hypertension. The damage of the brain is the main content. Melanocortin (MC) is becoming more and more concerned. MC plays the functions of regulating pigmentation, energy homeostasis, inflammatory control and immunoregulation through its different receptors in multiple organs. The research has found that the MC4 receptor (melanocortin 4 receptor, MC4R) agonists and their artificial agents have been found. Synthetic analogues can play a protective role in hemorrhagic shock, ischemic stroke and traumatic brain injury, and play a rapid and anti inflammatory role through the cholinergic anti-inflammatory pathway, but the specific mechanism is not clear. The purpose of this study is to explore whether MC can play a role in alleviating intestinal and brain dysfunction in secondary IAH and further explore. The mechanism is expected to provide a new strategy for the treatment of patients with clinical abdominal hypertension. In order to test the above hypothesis, this study first established the secondary IAH rat model. This model should be easy to operate, can be highly reproducible and can simulate the pathogenesis of IAH to a certain extent. The second, the observation is given RO27-3225 (MC4R agonist) can be reduced. The protective effect of the protective effect of light IAH on the intestinal damage and the effect of the protective effect. Finally, the effect of RO27-3225 on IAH induced intraperitoneal organ damage, brain injury, and the role of cholinergic signal pathway in it were judged. Method 1. the mean arterial pressure (mean AR) was induced by the method of blood release after femoral artery intubation. Terial pressure, MAP), 40mm Hg (maintain 120 min), 30mm Hg (maintain 90min) and 25mm Hg (maintain 60min), and then all merged (maintain 4) to establish a secondary rat model, use the dry wet ratio method to detect the water content of the intestine and brain tissue, and observe the intestinal morphology and analysis of the rat's intestines under the light microscope. The survival rate of the same group was selected from the IAH model of rats after shock, which could both cause obvious damage and guarantee survival rate for subsequent experiments. The following observation was made by.2. in the secondary IAH rat model (30mm Hg maintenance 90min combined with IAP=20mm Hg to maintain 4h), by observing the degree of change of MAP in rats; real-time PCR method was used. The changes in the whole intestinal wall inflammatory factors (TNF- - and IL-1 beta) were detected, the changes in the intestinal water content were detected by the dry wet ratio method, and the changes of the intestinal tissue in the rats were observed by the HE staining method, and the changes in the intestinal fatty acid binding protein were detected by the enzyme linked immunosorbent assay, and the isothiocyanate fluorescent protein was detected to reflect the intestinal tract. The key factors affecting the permeability of the intestinal tract, ROCK1, the level of myosin light chain phosphorylation, and the changes of the superoxide dismutase in the intestinal tract were detected by Western blot, and the effects of IAH on the intestinal damage were investigated. On the basis of this, the effect of RO27-3225 on the protection of intestinal tract was further observed,.3. In the secondary IAH rat model, the changes in the water content of the brain were detected by the dry wet ratio method, and the blood brain barrier permeability was detected by Evans blue exosmosis. The changes of the brain inflammatory factors (TNF- and IL-1 beta) were detected by real-time PCR and ELISA, and the water channel eggs were detected by enzyme linked immunosorbent assay and Western blot. The changes in the expression of white 4 and metal matrix proteinase 9 and the changes of the expression of glial cells by immunohistochemical method were used to investigate the effect of IAH on brain damage, and further to observe the effect of RO27-3225 on the protection of brain tissue. Results 1. the results were selected as the condition of MAP for 30mm Hg holding 90 min with IAP=20 mm Hg continuous 4 h. It can induce obvious histological damage in the intestinal tract of rats, which can lead to an obvious increase in the water content of the intestine, and ensure the survival rate to ensure the follow-up test. 2.RO27-3225 can stabilize the MAP changes to a certain extent, reduce the increase of the intestinal inflammatory factors (TNF- A and IL-1 beta) induced by IAH, and alleviate the ROCK 1 protein table in the intestinal tract caused by IAH. The level of light chain phosphorylation of myosin and myosin decreases, reduces the level of intestinal fatty acid binding protein, reduces the abnormal changes in intestinal edema, intestinal permeability and intestinal histology, and thus plays a role in alleviating the intestinal damage caused by IAH, and the protective effect can be treated by its specific blocker (HS024) and nicotinic acetylcholate. The alkali receptor antagonist (cisisan Daming) prevents.3.RO27-3225 from the brain edema caused by IAH, and reduces the expression of inflammatory factors, aquaporin 4 and metal matrix protease 9, which can play a role in alleviating brain damage in IAH, and this protective effect can be treated by its specific blocker (HS024) and nicotinic acetylcholine receptor antagonist (chloroisoyl). Sang Daming) prevented. But it did not affect the increase of the expression of glial cells induced by IAH. Conclusion 1.IAH can cause obvious edema and inflammatory reaction in the visceral organs of the abdominal cavity, and there is obvious histological damage. In addition, IAH can induce obvious inflammatory disease in the external organ - brain tissue, edema and damage of blood brain barrier integrity; 2. Selective MC4R agonist (RO27-3225) can stabilize the hemodynamics of the body, and by regulating the expression of ROCK1 and the level of myosin light chain phosphorylation to alleviate the increase in intestinal permeability and reduce the degree of inflammation, and effectively alleviate the intestinal damage induced by IAH. The protective effect may be through the cholinergic pathway; 3. Sexual MC4R agonists can reduce the destruction of blood brain barrier by reducing the expression of matrix metalloproteinase 9, reducing the degree of brain edema through the action of aquaporin 4, and alleviating the inflammatory response of brain tissue to effectively improve the brain tissue damage caused by IAH. This protective effect may be achieved through the bile alkali energy pathway. There was no obvious effect on the number of glial cells.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R641

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