發(fā)布時(shí)間：2018-05-15 10:25

  本文選題：燒傷 + 急性肺損傷�。� 參考：《第二軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：一、研究背景 嚴(yán)重?zé)齻麜?huì)導(dǎo)致機(jī)體內(nèi)環(huán)境發(fā)生劇烈變化。創(chuàng)傷應(yīng)激、熱力直接損傷、組織低灌注等因素會(huì)引起單核巨噬細(xì)胞、中性粒細(xì)胞、淋巴細(xì)胞、血小板、內(nèi)皮細(xì)胞等炎細(xì)胞活化，促炎性細(xì)胞因子表達(dá)增多；血管內(nèi)皮細(xì)胞和白細(xì)胞受到強(qiáng)烈刺激后粘附分子表達(dá)及活化增強(qiáng)，而且隨著兩者粘附的加強(qiáng)，激活的白細(xì)胞出現(xiàn)呼吸爆炸、脫顆粒從而釋放出大量蛋白溶酶、活性氧、花生四烯酸等代謝產(chǎn)物及促炎細(xì)胞因子，造成血管內(nèi)皮細(xì)胞和其他組織細(xì)胞的廣泛損傷。上述病理生理過程可概括為：嚴(yán)重?zé)齻傺准?xì)胞因子表達(dá)增多—→白細(xì)胞與血管內(nèi)皮細(xì)胞(endothelial cell，EC)粘附激活、釋放大量炎癥介質(zhì)—→失控的全身炎癥反應(yīng)綜合征（systemic inflammatory responsesyndrome, SIRS）。為了阻斷SIRS的發(fā)生和發(fā)展，近年來國內(nèi)外提出了一些針對(duì)單個(gè)細(xì)胞因子的治療，例如細(xì)胞因子的單克隆抗體和可溶性受體等，但在臨床試用均無明顯效果，甚至還有增加死亡的報(bào)道。分析其原因，主要是細(xì)胞因子及其代謝產(chǎn)物種類眾多，構(gòu)成了一個(gè)十分復(fù)雜的網(wǎng)絡(luò)，僅僅針對(duì)其中某一種或幾種進(jìn)行干預(yù)，往往達(dá)不到目的。但細(xì)胞因子作用于靶細(xì)胞時(shí)所必須經(jīng)過的受體后信號(hào)轉(zhuǎn)導(dǎo)通路則屈指可數(shù)，如果著重研究炎性介質(zhì)過度釋放的信號(hào)轉(zhuǎn)導(dǎo)機(jī)理，并對(duì)關(guān)鍵信號(hào)通路進(jìn)行調(diào)控，對(duì)于阻斷嚴(yán)重?zé)齻麜r(shí)失控性的全身炎癥反應(yīng)可能較之于針對(duì)某一單個(gè)炎癥介質(zhì)更為有效。 Toll樣受體（Toll-like receptors，TLRs）是一類病原分子識(shí)別受體家族，它不僅能識(shí)別病原體相關(guān)分子模式（PAMPs），其某些成員，尤其是TLR4也可識(shí)別無菌性損傷后釋放的眾多內(nèi)源性分子即損傷相關(guān)分子模式（DAMPs）。外源LPS或內(nèi)源性配體與TLR4結(jié)合后，活化MyD88和TRIF兩條信號(hào)途徑，前者活化NF-κB和MAPK信號(hào)通路，后者活化NF-κB和干擾素調(diào)節(jié)因子3信號(hào)通路。TLR4通過這些信號(hào)途徑誘導(dǎo)產(chǎn)生一系列的炎癥介質(zhì)包括細(xì)胞因子、趨化因子等，從而產(chǎn)生強(qiáng)有力的炎癥和免疫反應(yīng)。近年來研究表明，TLR4及其信號(hào)通路介導(dǎo)的天然免疫在感染和非感染性疾病引起的局部和全身炎癥反應(yīng)發(fā)生、發(fā)展的動(dòng)態(tài)過程中具有重要作用。 Bruton酪氨酸蛋白激酶（Bruton’s tyrosine kinase，Btk）是胞漿非受體酪氨酸蛋白激酶Tec家族成員之一，它是前B淋巴細(xì)胞發(fā)育所必需的。Btk的細(xì)胞表達(dá)譜較窄，僅限于髓系細(xì)胞，不表達(dá)于T淋巴細(xì)胞及組織漿細(xì)胞。Btk可增強(qiáng)TLR信號(hào)轉(zhuǎn)導(dǎo)并最終激活NF-κB和MAPKs信號(hào)通路，繼而啟動(dòng)炎性基因表達(dá)。研究表明，Btk基因突變或缺失的單核/巨噬細(xì)胞、肥大細(xì)胞和外周血單個(gè)核細(xì)胞對(duì)LPS刺激不敏感，TNF-α、IL-1β和iNOS表達(dá)明顯下降，進(jìn)一步研究發(fā)現(xiàn)Btk對(duì)LPS誘導(dǎo)的細(xì)胞因子表達(dá)的調(diào)控作用主要是通過NF-κB和p38激酶介導(dǎo)的。目前關(guān)于Btk信號(hào)通路與炎癥關(guān)系的研究仍多限于體外觀察，它在體內(nèi)炎癥反應(yīng)中的調(diào)控作用仍知之甚少。 臨床觀察表明，肺臟是燒傷后功能不全發(fā)生率最高和發(fā)生時(shí)間最早的器官。肺臟不僅是氣體交換的器官，也是一些細(xì)胞因子和激素產(chǎn)生和滅活的場(chǎng)所，加上肺臟本身在解剖學(xué)上相對(duì)較脆弱，易受到各種致病因素的打擊，因此肺臟是燒傷后炎性損害的主要靶器官之一。由于失控的炎癥反應(yīng)是ALI發(fā)病的病理生理學(xué)基礎(chǔ)，Btk作為眾多炎性介質(zhì)合成與釋放的重要上游信號(hào)轉(zhuǎn)導(dǎo)通路，可能在ALI的發(fā)生與發(fā)展過程中發(fā)揮了重要作用。因此，本課題定位于肺組織，研究Btk信號(hào)轉(zhuǎn)導(dǎo)通路與嚴(yán)重?zé)齻缙谘装Y調(diào)控的關(guān)系以及其與燒傷后肺臟損傷的關(guān)系。 二、研究目的 1.觀察Btk信號(hào)通路在嚴(yán)重?zé)齻∈蠓闻K中的表達(dá)及活化情況，初步探討B(tài)tk信號(hào)通路在嚴(yán)重?zé)齻蠹毙苑螕p傷發(fā)病及局部炎癥反應(yīng)中的作用。 2.觀察抑制Btk活化后，嚴(yán)重?zé)齻∈蠓谓M織局部信號(hào)蛋白的動(dòng)態(tài)變化情況，探討B(tài)tk在燒傷誘導(dǎo)的小鼠肺組織中MAPK和NF-κB及相關(guān)通路參與炎性肺損傷發(fā)病機(jī)制的可能效應(yīng)。 三、研究?jī)?nèi)容第一部分Btk激酶活化在燒傷后急性肺損傷發(fā)病中的作用 健康成年的雄性C57BL/6小鼠252只，隨機(jī)分為假燙對(duì)照組（C組）、燒傷組（S組），燒傷+LFM-A13組（L組）。各組小鼠均予備皮、麻醉處置，S組、L組小鼠后軀干置98℃沸水中12s，復(fù)制30%TBSA III°燙傷模型。L組在燒傷前1h及燒傷后6h，按4.2mg/kg的劑量腹腔內(nèi)注射LFM-A13，C、S組則予等量平衡液。按Parkland公式4ml/(kg*1%TBSA)計(jì)算總補(bǔ)液量，分別于傷后即刻、6h時(shí)相點(diǎn)分別腹腔注射總補(bǔ)液量50%的平衡液；C組不予燒傷及補(bǔ)液。各組于傷后即刻、0.5h、1h、3h、6h和12h共6個(gè)時(shí)相點(diǎn)，各取6只小鼠的肺臟以10％中性福爾馬林液固定待病理檢查；各組另取6只小鼠行下腔靜脈取血處死后取肺臟，1×PBS漂洗后液氮凍存。再于術(shù)后12h，各組另取6只小鼠行下腔靜脈取血處死后取肺臟，以濾紙吸干表面滲液及血跡后稱重；再另各取6只小鼠從頸外靜脈注入1%伊文思藍(lán)，1小時(shí)后處死并灌洗肺循環(huán)，取左下肺待用。而后，全細(xì)胞裂解法提取各標(biāo)本總蛋白后，Western blot法檢測(cè)各組各時(shí)間點(diǎn)肺組織中Btk蛋白、磷酸化Btk蛋白、Caspase-3和Bcl-2活性含量；采用Carraway雙盲病理評(píng)分，，評(píng)價(jià)各組小鼠肺損傷程度；免疫組織化學(xué)法對(duì)各組小鼠肺組織Btk蛋白進(jìn)行定位檢測(cè)；原位末端轉(zhuǎn)移酶標(biāo)記技術(shù)（TUNEL）檢測(cè)各組小鼠肺泡上皮細(xì)胞凋亡情況；肺干濕重比測(cè)定各組小鼠肺組織含水量；伊文氏藍(lán)比色法檢測(cè)各組小鼠肺微血管通透性。第二部分Btk激酶在燒傷后肺內(nèi)促炎性細(xì)胞因子表達(dá)和中性粒細(xì)胞浸潤(rùn)中的作用 采用第一部分?jǐn)⑹龅姆椒ㄖ谱鲃?dòng)物模型，在燒傷后12小時(shí)取材，處理標(biāo)本。采用酶聯(lián)免疫吸附試驗(yàn)測(cè)定各組小鼠血清TNF-α、IL-1β、IL-6、NO2-/NO3-蛋白含量；Trizol法提取肺組織總mRNA后，Real time PCR法分析TNF-α、IL-1β、IL-6、iNOS mRNA表達(dá)水平；測(cè)定肺組織髓過氧化物酶（MPO）活性。第三部分Btk激酶參與燒傷后肺炎性損傷的分子作用機(jī)制 采用第一部分?jǐn)⑹龅姆椒ㄖ谱鲃?dòng)物模型，在燒傷后即刻、0.5h、1h、3h、6h和12h共6個(gè)時(shí)相點(diǎn)，各組小鼠行下腔靜脈抽凈血處死，分離肺臟，1×PBS漂洗表面血跡后，無菌濾紙吸干殘液，液氮冰凍條件下研磨成粉狀，分裝于Eppendorf管，置-80℃冰箱保存。以全細(xì)胞裂解法提取各標(biāo)本總蛋白，Western blot法檢測(cè)各組各時(shí)間點(diǎn)的肺組織中磷酸化p38、磷酸化JNK、磷酸化ERK、IκBα、磷酸化IκBα蛋白的含量，探討B(tài)tk參與燒傷后肺炎性損傷的分子作用機(jī)理。 四、研究結(jié)果第一部分 燒傷組小鼠肺臟的病理評(píng)分在燒傷后6h至12h一直顯著高于假燙傷對(duì)照組，而燒傷+LFM-A13組小鼠肺臟的病理評(píng)分雖高于假燒傷對(duì)照組，但較燒傷組顯著降低。Western blot結(jié)果示，假燙對(duì)照組小鼠肺臟僅有少量Btk蛋白表達(dá)，而燒傷后30min Btk蛋白即有顯著增加，隨后仍持續(xù)增加，在6h達(dá)到高峰，并持續(xù)到12h；Btk磷酸化蛋白表達(dá)變化趨勢(shì)與其總蛋白表達(dá)一致。預(yù)先應(yīng)用LFM-A13后，燒傷后各時(shí)間點(diǎn)Btk磷酸化蛋白表達(dá)均較燒傷組顯著降低。免疫組化結(jié)果表明，Btk蛋白表達(dá)主要限于單核/巨噬細(xì)胞和中性粒細(xì)胞等浸潤(rùn)的炎性細(xì)胞。 原位末端轉(zhuǎn)移酶標(biāo)記技術(shù)（TUNEL）檢測(cè)及細(xì)胞凋亡指數(shù)（AI）顯示，假燙對(duì)照組基本未見凋亡細(xì)胞；燒傷組則凋亡最為明顯；燒傷+LFM-A13組可見凋亡，但較燒傷組明顯減少。Western blot結(jié)果提示，與假燙對(duì)照組比較，燒傷組小鼠肺組織內(nèi)凋亡關(guān)鍵蛋白活化型Caspase-3、抗凋亡關(guān)鍵蛋白Bcl-2表達(dá)均顯著增加；燒傷+LFM-A13組小鼠Caspase-3表達(dá)較燒傷組明顯下降，Bcl-2表達(dá)則較燒傷組更為顯著。 肺干濕重比及伊文氏藍(lán)比色法顯示，燒傷后12h時(shí)相點(diǎn)，燒傷組肺組織含水量及微血管通透性均較假燙對(duì)照組顯著增高，燒傷+LFM-A13組肺組織含水量及微血管通透性亦顯著高于假燙對(duì)照組，但較燒傷組顯著降低。 第二部分 酶聯(lián)免疫吸附試驗(yàn)（ELISA）結(jié)果示，燒傷后12h，燒傷組小鼠血清TNF-α、IL-1β、IL-6、NO2-/NO3-水平均顯著高于假燙對(duì)照組；燒傷+LFM-A13組小鼠血清TNF-α、IL-1β、IL-6、NO2-/NO3-水平亦顯著高于假燙對(duì)照組，但較燒傷組顯著降低。 燒傷組小鼠在燒傷后12h其肺組織TNF-α、IL-1β、IL-6和iNOS mRNA的表達(dá)量始終顯著高于假燙對(duì)照組；而燒傷+LFM-A13組小鼠肺組織TNF-α、IL-1β、IL-6和iNOS mRNA的表達(dá)水平雖仍明顯高于假燙對(duì)照組水平，但卻顯著低于燒傷組。 小鼠30％TBSAIII°燒傷后12小時(shí)，燒傷組小鼠肺組織的MPO活性顯著高于假燙對(duì)照組；燙傷+LFM-A13組MPO活性也顯著高于假燙對(duì)照組，但較燒傷組顯著降低。 第三部分 Western blot法檢測(cè)各時(shí)間點(diǎn)肺組織中磷酸化p38、磷酸化JNK、磷酸化ERK、IκBα、磷酸化IκBα的表達(dá)量并比較發(fā)現(xiàn)，在燒傷后30min，p38和ERK即發(fā)生明顯活化，而JNK在燒傷后3h才開始明顯活化；NF-κB在燒傷30min后即發(fā)生明顯活化，但其活化隨時(shí)間點(diǎn)推移逐漸減弱。而使用LFM-A13預(yù)干預(yù)后再給予燒傷刺激，與單純燒傷組比較發(fā)現(xiàn)，抑制Btk活化可顯著抑制燒傷后早期p38和NF-κB活化，但不影響JNK和ERK活化。 五、研究結(jié)論 （1） Btk特異性抑制劑可明顯抑制燒傷小鼠肺組織Btk活化，減輕Btk蛋白在單核/巨噬細(xì)胞及中性粒細(xì)胞等炎性細(xì)胞中的表達(dá)，從而降低肺微血管通透性，減輕肺組織水腫，減少中性粒細(xì)胞在肺組織內(nèi)的扣押；同時(shí)這種抑制效應(yīng)可明顯降低燒傷后相關(guān)促炎細(xì)胞因子的血清水平及其在肺組織內(nèi)的mRNA表達(dá)水平，從而有效減輕了燒傷后急性肺損傷。 （2）抑制Btk激酶活化可顯著抑制燒傷后早期p38和NF-κB活化，但不影響JNK和ERK活化，提示Btk在燒傷后急性肺損傷中的作用可能是通過下游p38和NF-κB信號(hào)通路活化介導(dǎo)的。
[Abstract]:First, research background
Severe burns can cause severe changes in the environment of the body. Trauma stress, direct thermal damage, and tissue low perfusion may cause the activation of mononuclear macrophages, neutrophils, lymphocytes, platelets, endothelial cells and other inflammatory cells, and increase the expression of inflammatory cytokines; vascular endothelial cells and leukocytes are strongly stimulated after the adhesion. The expression and activation of the attached molecules are enhanced, and as the adhesion strengthens, the activated white cells appear breathing explosion, degranulation so as to release a large number of proteolytic enzymes, active oxygen, peanut four enoic acid and other metabolites and pro-inflammatory cytokines, causing extensive damage to vascular endothelial cells and other histopathology cells. The above pathophysiological process can be generalized. It includes: Severe Burns - increased expression of proinflammatory cytokines - the adhesion and activation of white blood cells and vascular endothelial cells (endothelial cell, EC), releasing a large number of inflammatory mediators - systemic inflammatory responsesyndrome (SIRS). In order to block the occurrence and development of SIRS, it has been proposed at home and abroad in recent years. There are some treatments for single cytokine, such as monoclonal antibodies and soluble receptors of cytokines, but there are no obvious effects in clinical trials, even reports of increasing death. The reasons are mainly the numerous types of cytokines and their metabolites, which are made into a very complex network, only for one of them. One or several kinds of intervention often fail to reach the goal. But the postreceptor signal transduction pathway that the cytokine has to pass through the target cell is numbered. If the signal transduction mechanism of excessive release of inflammatory mediators is studied and the key signaling pathways are regulated, it is necessary to block out of control systemic inflammation in severe burns. The response may be more effective than targeting a single inflammatory mediator.
The Toll like receptor (Toll-like receptors, TLRs) is a class of pathogenic molecular recognition receptor family. It can not only identify the pathogen associated molecular model (PAMPs). Some of its members, especially TLR4, can also identify many endogenous molecules released after aseptic injury (DAMPs). Exogenous LPS or endogenous ligands are combined with TLR4. Activation of two signal pathways of MyD88 and TRIF, the former activates NF- kappa B and MAPK signaling pathway, and the latter activates NF- kappa B and interferon regulatory factor 3 signaling pathway.TLR4 through these signaling pathways to produce a series of inflammatory mediators, including cytokines, chemokines, etc., and thus produce strong inflammatory and immune responses. Recent studies have shown that Natural immunity mediated by TLR4 and its signaling pathway plays an important role in the development of local and systemic inflammatory reactions caused by infection and non infectious diseases.
Bruton tyrosine protein kinase (Bruton 's tyrosine kinase, Btk) is one of the members of the cytoplasmic non receptor tyrosine protein kinase Tec family. It is the narrow cell expression of the.Btk necessary for the development of the anterior B lymphocyte. It is limited to myeloid cells. The non expression of the T lymphocyte and the group of the plasma cells can enhance TLR signal transduction and eventually activate it. - kappa B and MAPKs signaling pathways, then starting inflammatory gene expression. Studies have shown that Btk gene mutations or missing mononuclear / macrophages, mast cells and peripheral blood mononuclear cells are insensitive to LPS stimulation, and the expression of TNF-, IL-1 beta and iNOS is significantly decreased. Further studies have found that Btk regulates the expression of LPS induced cytokines by Btk. NF- - kappa B and p38 kinases are mediated. The current study of the relationship between the Btk signaling pathway and inflammation is still limited to in vitro observation, and its role in the regulation of inflammation in the body is still poorly understood.
The clinical observation shows that the lung is the organ that has the highest incidence of dysfunction after burn and the earliest time of occurrence. The lung is not only an organ of gas exchange, but also a place for producing and inactivating some cytokines and hormones, and the lung itself is relatively vulnerable to anatomy and is vulnerable to various pathogenic factors, so the lungs are burned. One of the major target organs for inflammatory damage is the pathophysiological basis of ALI, which is an important upstream signal transduction pathway for the synthesis and release of many inflammatory mediators, which may play an important role in the development and development of ALI. Therefore, this topic is located in the lung tissue and studies the Btk signal transduction. The relationship between pathway and inflammation in early stage of severe burn injury and its relationship with lung injury after burn injury.
Two, the purpose of the study
1. to observe the expression and activation of Btk signaling pathway in the lungs of severely burned mice, and to explore the role of Btk signaling pathway in the pathogenesis of acute lung injury and local inflammatory response after severe burn.
2. to observe the dynamic changes of local signal protein in the lung tissue of severely burned mice after the inhibition of Btk activation, and to explore the possible effect of Btk on the pathogenesis of inflammatory lung injury induced by MAPK and NF- kappa B in the lung tissue induced by burn.
Three. Part 1: role of Btk kinase activation in the pathogenesis of acute lung injury after burn injury
252 healthy adult male C57BL/6 mice were randomly divided into the false ironing control group (group C), the burn group (group S) and the burn +LFM-A13 group (group L). The mice in each group were all given skin preparation, anaesthesia, and S group. The trunk of the L group was placed in the boiling water of 12s in the trunk of the mice, and the.L group in the 30%TBSA III degree scald model was injected into the abdominal cavity before and after the burn. FM-A13, C, and S group were given equal amount of balance fluid. According to the Parkland formula 4ml/ (kg*1%TBSA), the total fluid amount was calculated, and the balance solution was injected respectively after the injury, and the phase point of 6h was 50%, respectively. The C group did not have the burn and rehydration. Each group was immediately after the injury, 0.5h, 1H, 3h, 6h and 6 phases, each of the lungs of 6 mice was 10% neutral formma. 6 mice in each group were taken blood from the inferior vena cava to get the lungs and 1 x PBS after 1 x rinse. Then after the operation, 6 mice were taken from the inferior vena cava and took the blood to get the lungs, then the filter paper was used to dry the surface seepage and blood, and then the other 6 mice were injected from the external jugular vein of 1% Evans. After 1 hours, death and lavage of lung circulation and left lower lung were used. After the total cell lysis method was used to extract the total protein, Btk protein, phosphorylated Btk protein, Caspase-3 and Bcl-2 activity in each time point of lung tissue were detected by Western blot method, and the degree of lung injury in each group was evaluated by Carraway double blind pathological score. The pestilence histochemical method was used to detect the Btk protein in lung tissue of mice in each group; in situ TDT labeling technique (TUNEL) was used to detect the apoptosis of alveolar epithelial cells in each group; the water content of lung tissue in each group was measured by the ratio of dry wet weight to the lung tissue in each group; the second part of Btk kinase was detected by Evans blue colorimetry. Role of proinflammatory cytokines and neutrophil infiltration in lung after burn injury
The animal model was made in the first part of the animal model, and the specimens were obtained 12 hours after the burn. The serum TNF- a, IL-1 beta, IL-6 and NO2-/NO3- protein were measured by enzyme linked immunosorbent assay. After the Trizol method was used to extract the total mRNA of lung tissue, the Real time PCR method was used to analyze TNF- a, IL-1 beta, IL-6, and the expression level. Tissue myeloperoxidase (MPO) activity. The third part of Btk kinase is involved in the molecular mechanism of pneumonia after burns.
The animal model was made in the first part of the method. After the burn, 0.5h, 1H, 3h, 6h and 12h were 6 phase points. All mice were killed in the inferior vena cava and separated from the lungs. After 1 x PBS rinse the surface of the surface blood, the sterile filter paper sucked the residual liquid, and the liquid nitrogen was grinded into powder under the freezing condition. It was stored in the Eppendorf tube and stored in -80 centigrade refrigerator. Total cell protein was extracted with total cell lysis. Western blot method was used to detect the phosphorylated p38, phosphorylated JNK, phosphorylated ERK, I kappa B alpha, and I kappa B alpha protein in the lung tissue of each time point, and to explore the molecular mechanism of Btk involved in the injury of pneumonia after burn.
Four, the first part of the study
The pathological score of lung in the burn group was significantly higher than that of the control group in the 6h to 12h after burn, while the pathological score of lung in the +LFM-A13 group was higher than that of the false burn control group, but the result of the decrease of.Western blot in the burn group showed that only a small amount of Btk protein was expressed in the lung of the sham control group and the 30min Btk eggs after the burn. There was a significant increase in white, and then continued to increase, reaching the peak at 6h and continuing to 12h; the expression of Btk phosphorylation protein was consistent with the total protein expression. After the use of LFM-A13, the expression of Btk phosphorylated protein in every time point after burn was significantly lower than that in the burn group. The immunohistochemical results showed that the expression of Btk protein was mainly limited to mononuclear / mononuclear cells. Inflammatory cells infiltrated by macrophages and neutrophils.
In situ end transferase labeling technique (TUNEL) detection and apoptosis index (AI) showed that no apoptotic cells were found in the control group and apoptosis was the most obvious in the burn group, and apoptosis in the burn group +LFM-A13 group, but the results of.Western blot in the burn group showed that the apoptosis in the lung tissue of the burn group was compared with that of the sham control group. The expression of key protein activated Caspase-3 and the expression of key protein Bcl-2 were significantly increased, and the expression of Caspase-3 in the burn +LFM-A13 group was significantly lower than that in the burn group, and the expression of Bcl-2 was more significant than that in the burn group.
The lung dry and wet weight ratio and the Evans blue colorimetric method showed that the water content and microvascular permeability of the lung tissue in the burn group were significantly higher than those in the control group after 12h, and the water content and microvascular permeability of the lung tissue in the +LFM-A13 group were significantly higher than those in the sham control group, but it was significantly lower than that in the burn group.
The second part
The results of enzyme linked immunosorbent assay (ELISA) showed that the levels of serum TNF- a, IL-1 beta, IL-6 and NO2-/NO3- in burned mice were significantly higher than those in the sham control group after burn 12h, and the serum TNF- a, IL-1 beta, IL-6 and NO2-/NO3- in the +LFM-A13 group were significantly higher than those in the sham control group, but significantly lower than those in the burn group.
The expression of TNF- alpha, IL-1 beta, IL-6 and iNOS mRNA in the lung tissue of burn mice was always significantly higher than that in the control group after burn, while the expression level of TNF- a, IL-1 beta, IL-6 and iNOS mRNA in the lung tissue of the burn +LFM-A13 group was still significantly higher than that of the false iron control group, but it was significantly lower than that in the burn group, but it was significantly lower than that in the burn group.
12 hours after 30%TBSAIII degree burn in mice, the MPO activity of lung tissue in the burn group was significantly higher than that in the sham control group, and the activity of MPO in the scalded +LFM-A13 group was significantly higher than that in the sham control group, but it was significantly lower than that in the burn group.
The third part
Western blot method was used to detect phosphorylated p38, phosphorylated JNK, phosphorylated ERK, I kappa B alpha, and phosphorylated I kappa B alpha, and found that 30min, p38 and ERK began to activate obviously after burn, and the activation was observed after burn injury, but its activation followed the time point. The shift gradually weakened. And the LFM-A13 predry prognosis was given to the burn stimulation. Compared with the simple burn group, the inhibition of Btk activation could significantly inhibit the activation of p38 and NF- kappa B in the early post burn, but did not affect the activation of JNK and ERK.
Five, the conclusion of the study
(1) Btk specific inhibitors can significantly inhibit the activation of Btk in lung tissue of burned mice, reduce the expression of Btk protein in mononuclear / macrophage and neutrophils, thus reduce pulmonary microvascular permeability, reduce pulmonary edema and reduce the seizure of neutrophils in the lung tissue, and this inhibitory effect can be significantly reduced. The serum level of proinflammatory cytokines and the expression level of mRNA in lung tissue after burn can effectively alleviate acute lung injury after burns.
(2) inhibition of Btk kinase activation can significantly inhibit the activation of p38 and NF- kappa B in the early post burn, but does not affect the activation of JNK and ERK, suggesting that the role of Btk in acute lung injury after burn may be mediated by the activation of the downstream p38 and NF- kappa B signaling pathway.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R644;R-332

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