本文選題：失血性休克 + 急性肺損傷��； 參考：《河北北方學(xué)院》2017年碩士論文

【摘要】：急性肺損傷(acute lung injury,ALI)是由多種致病因素造成的肺實(shí)質(zhì)細(xì)胞出現(xiàn)嚴(yán)重炎癥反應(yīng),導(dǎo)致肺通透性增高、發(fā)生急性肺水腫的結(jié)果。由于失血性休克引起的微循環(huán)障礙以及肺的血流動(dòng)力學(xué)特點(diǎn),使得失血性休克患者最易發(fā)生ALI,可迅速發(fā)展為急性呼吸窘迫綜合征(acute respiratory distress syndrome,ARDS),嚴(yán)重危及患者生命。我室前期研究發(fā)現(xiàn),減少腸淋巴液回流,可減輕肺損傷,在降低肺組織氧化應(yīng)激的同時(shí),降低了肺組織Toll樣受體2(toll-like receptor 2,TLR2)、TLR4的表達(dá)以及高遷移率族蛋白B1(high-mobility group box1,HMGB1)的表達(dá)。研究表明,失血性休克后,活性氧(reactive oxygen species,ROS)大量產(chǎn)生與釋放,通過激發(fā)組織機(jī)體的炎性反應(yīng)、介導(dǎo)細(xì)胞凋亡及增加血管通透性等方面導(dǎo)致ALI。但失血性休克后腸淋巴液(post-hemorrhagic shock mesenteric lymph,PHSML)介導(dǎo)ALI過程中是否有ROS的參與,尚不清楚。減少PHSML回流是否可降低肺組織ROS的產(chǎn)生與釋放,PHSML介導(dǎo)肺組織ROS的產(chǎn)生與釋放是否受TLR2、TRL4、HMGB1的影響(和/或影響TLR2、TLR4、HMGB1的表達(dá))?有待研究。因此,本文在前期研究基礎(chǔ)上,采用失血性休克小鼠模型,觀察PHSML引流對失血性休克小鼠肺組織結(jié)構(gòu)以及ROS含量變化的影響,觀察靜脈回輸PHSML是否引起小鼠(野生型、TLR2-/-、TLR4-/-)肺組織結(jié)構(gòu)損傷、ROS產(chǎn)生與釋放及TLR2、TLR4分子表達(dá),并進(jìn)一步從細(xì)胞層面觀察ROS抑制劑(NAC)、TLR2及TLR4封閉性抗體處理小鼠肺微血管內(nèi)皮細(xì)胞(pulmonary microvascular endothelial cells,PMVECs)后,PHSML對PMVECs產(chǎn)生ROS的影響,從而驗(yàn)證PHSML是肺組織ROS產(chǎn)生增多的作用因素之一,該作用是通過TLR2、TLR4、HMGB1介導(dǎo)的。首先,將野生型(WT)C57BL/6J小鼠18只隨機(jī)均分為假手術(shù)組(Sham)、失血性休克組(Shock)、休克+引流組(Shock+drainage)組;常規(guī)方法建立失血性休克模型,觀察PHSML對失血性休克小鼠肺組織形態(tài)與ROS含量的作用。結(jié)果顯示,Sham組小鼠肺組織結(jié)構(gòu)基本正常;Shock組小鼠肺組織嚴(yán)重破壞,間隔增寬,多見炎性滲出及出血,肺泡腔大量融合;Shock+drainage組小鼠肺組織損傷較輕。同時(shí)可見,失血性休克引起了小鼠肺組織ROS顯著增高,PHSML引流顯著降低了Shock組小鼠肺組織ROS含量。隨后,應(yīng)用TLR2-/-、TLR4-/-小鼠各12只,均分為Sham、Shock組,常規(guī)方法建立失血性休克模型,觀察TLR2、TLR4基因敲除對失血性休克小鼠肺組織形態(tài)與ROS含量的作用。結(jié)果顯示,TLR2-/-、TLR4-/-的Shock組小鼠肺組織肺間隔炎性物質(zhì)滲出,間隔增寬,肺泡部分融合,但程度較WT小鼠低,且肺組織ROS含量均顯著低于WTShock組小鼠。然后,應(yīng)用50只WT小鼠,引流正常淋巴液(normal mesenteric lymph,NML)和PHSML(與前述相同的失血性休克模型),分別靜脈輸入至WT、TLR2-/-、TLR4-/-小鼠,每組6只,觀察靜脈輸入PHSML對各型小鼠肺組織形態(tài)、ROS含量以及TLR2、TLR4 mRNA表達(dá)的影響。結(jié)果顯示,靜脈輸入NML對WT、TLR2-/-、TLR4-/-小鼠肺組織結(jié)構(gòu)無明顯影響,靜脈輸入PHSML引起了WT正常小鼠出現(xiàn)了比較嚴(yán)重的肺組織損傷,而靜脈輸入PHSML對TLR2-/-、TLR4-/-小鼠肺組織損傷的程度較輕。同時(shí)可見,靜脈輸入PHSML引起了WT正常小鼠肺組織ROS含量以及TLR2、TLR4的mRNA表達(dá)顯著高于輸入NML組;靜脈輸入PHSML至TLR2-/-、TLR4-/-小鼠的肺組織ROS含量顯著低于WT小鼠,高于輸入NML的同型小鼠;靜脈輸入PHSML至TLR4-/-小鼠肺組織的TLR2 mRNA表達(dá)顯著高于輸入NML的TLR4-/-小鼠以及輸入PHSML的WT小鼠;同樣,靜脈輸入PHSML至TLR2-/-小鼠肺組織的TLR4 mRNA表達(dá)顯著高于輸入NML的TLR2-/-小鼠以及輸入PHSML的WT小鼠。為了進(jìn)一步驗(yàn)證ROS在PHSML介導(dǎo)ALI中的作用及其與TLR2、TLR4的關(guān)系,本文以小鼠肺微血管內(nèi)皮細(xì)胞(PMVECs)作為研究對象,觀察了ROS特異性抑制劑NAC、TLR2和TLR4封閉性抗體對PHSML損傷PMVECs以及促進(jìn)PMVECs產(chǎn)生ROS的作用。結(jié)果顯示,PHSML引起了PMVECs損傷、ROS產(chǎn)生增多,NAC、TLR2和TLR4封閉性抗體均減輕了PHSML導(dǎo)致的PMVECs結(jié)構(gòu)損傷,抑制了PHSML增加PMVECs產(chǎn)生ROS的作用。此外,針對HMGB1在PHSML介導(dǎo)ALI中的作用,本文進(jìn)一步觀察了正常小鼠腹腔注射HMGB1抑制劑甘草酸(Glycyrrhizic acid,GL)處理后,靜脈輸入PHSML后肺組織形態(tài)與ROS含量的變化。結(jié)果顯示,GL預(yù)先處理顯著降低了靜脈輸入PHSML損傷正常小鼠肺組織、增加ROS產(chǎn)生的作用。說明HMGB1參與了PHSML介導(dǎo)ALI的ROS機(jī)制。上述研究表明,PHSML引起肺組織ROS的過度產(chǎn)生是失血性休克ALI的重要機(jī)制之一;PHSML介導(dǎo)肺組織ROS過度產(chǎn)生與釋放的機(jī)制與TLR2、TLR4、HMGB1有關(guān)。以PHSML、ROS為干預(yù)靶點(diǎn),對于防治重癥休克導(dǎo)致的ALI具有積極的作用。
[Abstract]:Acute lung injury (acute lung, injury, ALI) is caused by various pathogenic factors of lung parenchyma cells appeared severe inflammation, leading to pulmonary permeability, acute pulmonary edema results. The microcirculation in hemorrhagic shock caused by pulmonary and hemodynamic characteristics of the patients with hemorrhagic shock were most prone to ALI, can be quickly for the development of acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS), seriously endanger the lives of patients. Our previous study found that reducing the intestinal lymph circulation, can reduce lung injury, reduce oxidative stress in lung tissue and reduce the Toll like receptor in lung tissue of 2 (Toll-like receptor 2, TLR2, TLR4) the expression of high mobility group protein B1 (high-mobility group box1, HMGB1) expression. The results show that after hemorrhagic shock, reactive oxygen species (reactive oxygen, species, ROS) a large number of production and release, The inflammatory reaction to stimulate body tissue, mediated cell apoptosis and increased vascular permeability in hemorrhagic shock rats but ALI. (post-hemorrhagic shock mesenteric lymph, lymph PHSML) mediated whether ALI participates in the process of ROS, is not clear. Whether PHSML can reduce reflux reduced production and release of ROS in lung tissue. The production and release of PHSML mediated by ROS in lung tissue by TLR2, TRL4, HMGB1 (the effect of expression and / or effects of TLR2, TLR4, HMGB1)? To study. Therefore, this paper based on the previous studies, the hemorrhagic shock model in mice, observe the PHSML drainage on the effect of hemorrhagic shock in the lung tissue of mice the structure and the change of the content of ROS, observe the intravenous infusion of PHSML can cause mice (wild type, TLR2-/-, TLR4-/-) of lung tissue damage, ROS release and TLR2, TLR4 expression, and further from the cellular level observation ROS inhibitor (NAC), TLR2 and TLR4 blocking antibody treatment of mouse pulmonary microvascular endothelial cells (pulmonary microvascular endothelial cells, PMVECs), PHSML ROS effect on PMVECs, which validates that PHSML is one of ROS in lung tissue increased the role of factors, the effect is through TLR2, TLR4, HMGB1 mediated. First of all, the wild type (WT) 18 C57BL/6J mice were randomly divided into sham operation group (Sham), hemorrhagic shock group (Shock), the shock + drainage group (group Shock+drainage); conventional method to establish the model of hemorrhagic shock, to observe the effects of PHSML on hemorrhagic shock in mice lung tissue morphology and ROS content. Show, Sham group of mice lung tissue structure was normal; lung tissue of Shock group were severely damaged, septum, rare inflammatory exudation and hemorrhage, alveolar fusion; Shock+drainage group of lung tissue injury in mice. At the same time less visible, hemorrhagic Hugh G caused the lung tissue of mice ROS significantly increased, PHSML drainage decreased the content of ROS in group Shock, the lung tissue of mice. Then, the application of TLR2-/- in TLR4-/- mice, 12 rats each, divided into Sham, Shock group, the conventional method to establish the model of hemorrhagic shock, observation of TLR2, TLR4 gene knockout on hemorrhagic shock in mice lung tissue with the content of ROS. The results showed that TLR2-/-, TLR4-/- in Shock group lung interval inflammatory substance exudation, alveolar septum, partial fusion, but more WT mice is low, and the content of ROS in lung tissue were significantly lower than those in WTShock group. Then, the application of 50 WT mice (normal lymph drainage. Normal mesenteric lymph, NML) and PHSML (hemorrhagic shock model with the same), respectively, intravenous to WT, TLR2-/-, TLR4-/- mice, 6 rats in each group. The observation of intravenous infusion of PHSML of various types of mouse lung tissue morphology, content of ROS, TLR2, TLR4 Effect of mRNA expression. The results showed that intravenous infusion of NML of WT, TLR2-/-, TLR4-/- had no effect on lung tissue structure in mice, intravenous infusion of PHSML caused by WT in normal mice appeared more serious lung injury, and intravenous infusion of PHSML of TLR2-/- and TLR4-/- in mouse lung tissue injury to a lesser degree. At the same time, visible, vein enter the PHSML WT in the lung tissue of normal mice caused by the content of ROS, TLR2, TLR4 mRNA expression was significantly higher than that of the input NML group; intravenous infusion of PHSML to TLR2-/- and ROS in lung tissue of TLR4-/- mice was significantly lower than that in WT mice, the same type of mice is higher than the input NML; intravenous infusion of PHSML into TLR4-/- mice lung tissue TLR2 mRNA expression was significantly higher than that of the input of the NML input PHSML TLR4-/- mice and WT mice; similarly, intravenous infusion of PHSML and TLR2-/- in lung tissue of mice TLR4 mRNA expression was significantly higher than that of the input NML TLR2-/- mice and PHSML input WT mice. In order to verify the ROS in PHSML mediated ALI and its interaction with TLR2, TLR4, the mouse pulmonary microvascular endothelial cells (PMVECs) as the research object, the effect of ROS specific inhibitor of NAC, TLR2 and TLR4 blocking antibody effects on PHSML and PMVECs to promote the production of PMVECs ROS. The results showed that PHSML caused PMVECs damage, increased ROS production, NAC, TLR2 and TLR4 blocking antibody can reduce the damage caused by PMVECs PHSML, PHSML ROS inhibited the increase of PMVECs effect. In addition, the HMGB1 in PHSML mediated ALI effect, we further observed in normal mice by intraperitoneal injection the inhibitor of HMGB1 (Glycyrrhizic acid GL, glycyrrhizic acid) after treatment, the morphological changes of lung tissue and ROS content after intravenous infusion of PHSML. The results showed that GL pretreatment significantly decreased the intravenous infusion of PHSML injury of the normal lung tissues of mice, The increase of ROS production. It indicated that HMGB1 is involved in ROS mechanism mediated by PHSML ALI. The results of the study showed that PHSML induced overproduction of ROS in lung tissue is one of the important mechanisms of hemorrhagic shock ALI; PHSML mediated lung ROS overproduction and release mechanism and TLR2, TLR4, HMGB1. PHSML have to. ROS is the target for intervention, plays a positive role in the prevention and treatment of severe shock caused ALI.

【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】：2017
【分類號(hào)】：R563.8

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 牛春雨;趙自剛;張玉平;杜舒婷;常海峰;張揚(yáng);閆斌;尚金星;陳錦霞;;淋巴液引流對創(chuàng)傷失血性休克大鼠多器官損傷的影響[J];中華創(chuàng)傷雜志;2010年12期

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本文編號(hào)：1763754