Bruton酪氨酸激酶在严重烧伤小鼠急性肺损伤发病中的作用和机制

发布时间：2018-05-15 10:25

本文选题：烧伤 + 急性肺损伤　；参考：《第二军医大学》2012年硕士论文

【摘要】：一、研究背景严重烧伤会导致机体内环境发生剧烈变化。创伤应激、热力直接损伤、组织低灌注等因素会引起单核巨噬细胞、中性粒细胞、淋巴细胞、血小板、内皮细胞等炎细胞活化，促炎性细胞因子表达增多；血管内皮细胞和白细胞受到强烈刺激后粘附分子表达及活化增强，而且随着两者粘附的加强，激活的白细胞出现呼吸爆炸、脱颗粒从而释放出大量蛋白溶酶、活性氧、花生四烯酸等代谢产物及促炎细胞因子，造成血管内皮细胞和其他组织细胞的广泛损伤。上述病理生理过程可概括为：严重烧伤—→促炎细胞因子表达增多—→白细胞与血管内皮细胞(endothelial cell，EC)粘附激活、释放大量炎症介质—→失控的全身炎症反应综合征（systemic inflammatory responsesyndrome, SIRS）。为了阻断SIRS的发生和发展，近年来国内外提出了一些针对单个细胞因子的治疗，例如细胞因子的单克隆抗体和可溶性受体等，但在临床试用均无明显效果，甚至还有增加死亡的报道。分析其原因，主要是细胞因子及其代谢产物种类众多，构成了一个十分复杂的网络，仅仅针对其中某一种或几种进行干预，往往达不到目的。但细胞因子作用于靶细胞时所必须经过的受体后信号转导通路则屈指可数，如果着重研究炎性介质过度释放的信号转导机理，并对关键信号通路进行调控，对于阻断严重烧伤时失控性的全身炎症反应可能较之于针对某一单个炎症介质更为有效。 Toll样受体（Toll-like receptors，TLRs）是一类病原分子识别受体家族，它不仅能识别病原体相关分子模式（PAMPs），其某些成员，尤其是TLR4也可识别无菌性损伤后释放的众多内源性分子即损伤相关分子模式（DAMPs）。外源LPS或内源性配体与TLR4结合后，活化MyD88和TRIF两条信号途径，前者活化NF-κB和MAPK信号通路，后者活化NF-κB和干扰素调节因子3信号通路。TLR4通过这些信号途径诱导产生一系列的炎症介质包括细胞因子、趋化因子等，从而产生强有力的炎症和免疫反应。近年来研究表明，TLR4及其信号通路介导的天然免疫在感染和非感染性疾病引起的局部和全身炎症反应发生、发展的动态过程中具有重要作用。 Bruton酪氨酸蛋白激酶（Bruton’s tyrosine kinase，Btk）是胞浆非受体酪氨酸蛋白激酶Tec家族成员之一，它是前B淋巴细胞发育所必需的。Btk的细胞表达谱较窄，仅限于髓系细胞，不表达于T淋巴细胞及组织浆细胞。Btk可增强TLR信号转导并最终激活NF-κB和MAPKs信号通路，继而启动炎性基因表达。研究表明，Btk基因突变或缺失的单核/巨噬细胞、肥大细胞和外周血单个核细胞对LPS刺激不敏感，TNF-α、IL-1β和iNOS表达明显下降，进一步研究发现Btk对LPS诱导的细胞因子表达的调控作用主要是通过NF-κB和p38激酶介导的。目前关于Btk信号通路与炎症关系的研究仍多限于体外观察，它在体内炎症反应中的调控作用仍知之甚少。临床观察表明，肺脏是烧伤后功能不全发生率最高和发生时间最早的器官。肺脏不仅是气体交换的器官，也是一些细胞因子和激素产生和灭活的场所，加上肺脏本身在解剖学上相对较脆弱，易受到各种致病因素的打击，因此肺脏是烧伤后炎性损害的主要靶器官之一。由于失控的炎症反应是ALI发病的病理生理学基础，Btk作为众多炎性介质合成与释放的重要上游信号转导通路，可能在ALI的发生与发展过程中发挥了重要作用。因此，本课题定位于肺组织，研究Btk信号转导通路与严重烧伤早期炎症调控的关系以及其与烧伤后肺脏损伤的关系。二、研究目的 1.观察Btk信号通路在严重烧伤小鼠肺脏中的表达及活化情况，初步探讨Btk信号通路在严重烧伤后急性肺损伤发病及局部炎症反应中的作用。 2.观察抑制Btk活化后，严重烧伤小鼠肺组织局部信号蛋白的动态变化情况，探讨Btk在烧伤诱导的小鼠肺组织中MAPK和NF-κB及相关通路参与炎性肺损伤发病机制的可能效应。三、研究内容第一部分Btk激酶活化在烧伤后急性肺损伤发病中的作用健康成年的雄性C57BL/6小鼠252只，随机分为假烫对照组（C组）、烧伤组（S组），烧伤+LFM-A13组（L组）。各组小鼠均予备皮、麻醉处置，S组、L组小鼠后躯干置98℃沸水中12s，复制30%TBSA III°烫伤模型。L组在烧伤前1h及烧伤后6h，按4.2mg/kg的剂量腹腔内注射LFM-A13，C、S组则予等量平衡液。按Parkland公式4ml/(kg*1%TBSA)计算总补液量，分别于伤后即刻、6h时相点分别腹腔注射总补液量50%的平衡液；C组不予烧伤及补液。各组于伤后即刻、0.5h、1h、3h、6h和12h共6个时相点，各取6只小鼠的肺脏以10％中性福尔马林液固定待病理检查；各组另取6只小鼠行下腔静脉取血处死后取肺脏，1×PBS漂洗后液氮冻存。再于术后12h，各组另取6只小鼠行下腔静脉取血处死后取肺脏，以滤纸吸干表面渗液及血迹后称重；再另各取6只小鼠从颈外静脉注入1%伊文思蓝，1小时后处死并灌洗肺循环，取左下肺待用。而后，全细胞裂解法提取各标本总蛋白后，Western blot法检测各组各时间点肺组织中Btk蛋白、磷酸化Btk蛋白、Caspase-3和Bcl-2活性含量；采用Carraway双盲病理评分，，评价各组小鼠肺损伤程度；免疫组织化学法对各组小鼠肺组织Btk蛋白进行定位检测；原位末端转移酶标记技术（TUNEL）检测各组小鼠肺泡上皮细胞凋亡情况；肺干湿重比测定各组小鼠肺组织含水量；伊文氏蓝比色法检测各组小鼠肺微血管通透性。第二部分Btk激酶在烧伤后肺内促炎性细胞因子表达和中性粒细胞浸润中的作用采用第一部分叙述的方法制作动物模型，在烧伤后12小时取材，处理标本。采用酶联免疫吸附试验测定各组小鼠血清TNF-α、IL-1β、IL-6、NO2-/NO3-蛋白含量；Trizol法提取肺组织总mRNA后，Real time PCR法分析TNF-α、IL-1β、IL-6、iNOS mRNA表达水平；测定肺组织髓过氧化物酶（MPO）活性。第三部分Btk激酶参与烧伤后肺炎性损伤的分子作用机制采用第一部分叙述的方法制作动物模型，在烧伤后即刻、0.5h、1h、3h、6h和12h共6个时相点，各组小鼠行下腔静脉抽净血处死，分离肺脏，1×PBS漂洗表面血迹后，无菌滤纸吸干残液，液氮冰冻条件下研磨成粉状，分装于Eppendorf管，置-80℃冰箱保存。以全细胞裂解法提取各标本总蛋白，Western blot法检测各组各时间点的肺组织中磷酸化p38、磷酸化JNK、磷酸化ERK、IκBα、磷酸化IκBα蛋白的含量，探讨Btk参与烧伤后肺炎性损伤的分子作用机理。四、研究结果第一部分烧伤组小鼠肺脏的病理评分在烧伤后6h至12h一直显著高于假烫伤对照组，而烧伤+LFM-A13组小鼠肺脏的病理评分虽高于假烧伤对照组，但较烧伤组显著降低。Western blot结果示，假烫对照组小鼠肺脏仅有少量Btk蛋白表达，而烧伤后30min Btk蛋白即有显著增加，随后仍持续增加，在6h达到高峰，并持续到12h；Btk磷酸化蛋白表达变化趋势与其总蛋白表达一致。预先应用LFM-A13后，烧伤后各时间点Btk磷酸化蛋白表达均较烧伤组显著降低。免疫组化结果表明，Btk蛋白表达主要限于单核/巨噬细胞和中性粒细胞等浸润的炎性细胞。原位末端转移酶标记技术（TUNEL）检测及细胞凋亡指数（AI）显示，假烫对照组基本未见凋亡细胞；烧伤组则凋亡最为明显；烧伤+LFM-A13组可见凋亡，但较烧伤组明显减少。Western blot结果提示，与假烫对照组比较，烧伤组小鼠肺组织内凋亡关键蛋白活化型Caspase-3、抗凋亡关键蛋白Bcl-2表达均显著增加；烧伤+LFM-A13组小鼠Caspase-3表达较烧伤组明显下降，Bcl-2表达则较烧伤组更为显著。肺干湿重比及伊文氏蓝比色法显示，烧伤后12h时相点，烧伤组肺组织含水量及微血管通透性均较假烫对照组显著增高，烧伤+LFM-A13组肺组织含水量及微血管通透性亦显著高于假烫对照组，但较烧伤组显著降低。第二部分酶联免疫吸附试验（ELISA）结果示，烧伤后12h，烧伤组小鼠血清TNF-α、IL-1β、IL-6、NO2-/NO3-水平均显著高于假烫对照组；烧伤+LFM-A13组小鼠血清TNF-α、IL-1β、IL-6、NO2-/NO3-水平亦显著高于假烫对照组，但较烧伤组显著降低。烧伤组小鼠在烧伤后12h其肺组织TNF-α、IL-1β、IL-6和iNOS mRNA的表达量始终显著高于假烫对照组；而烧伤+LFM-A13组小鼠肺组织TNF-α、IL-1β、IL-6和iNOS mRNA的表达水平虽仍明显高于假烫对照组水平，但却显著低于烧伤组。小鼠30％TBSAIII°烧伤后12小时，烧伤组小鼠肺组织的MPO活性显著高于假烫对照组；烫伤+LFM-A13组MPO活性也显著高于假烫对照组，但较烧伤组显著降低。第三部分 Western blot法检测各时间点肺组织中磷酸化p38、磷酸化JNK、磷酸化ERK、IκBα、磷酸化IκBα的表达量并比较发现，在烧伤后30min，p38和ERK即发生明显活化，而JNK在烧伤后3h才开始明显活化；NF-κB在烧伤30min后即发生明显活化，但其活化随时间点推移逐渐减弱。而使用LFM-A13预干预后再给予烧伤刺激，与单纯烧伤组比较发现，抑制Btk活化可显著抑制烧伤后早期p38和NF-κB活化，但不影响JNK和ERK活化。五、研究结论（1） Btk特异性抑制剂可明显抑制烧伤小鼠肺组织Btk活化，减轻Btk蛋白在单核/巨噬细胞及中性粒细胞等炎性细胞中的表达，从而降低肺微血管通透性，减轻肺组织水肿，减少中性粒细胞在肺组织内的扣押；同时这种抑制效应可明显降低烧伤后相关促炎细胞因子的血清水平及其在肺组织内的mRNA表达水平，从而有效减轻了烧伤后急性肺损伤。（2）抑制Btk激酶活化可显著抑制烧伤后早期p38和NF-κB活化，但不影响JNK和ERK活化，提示Btk在烧伤后急性肺损伤中的作用可能是通过下游p38和NF-κB信号通路活化介导的。
[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.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R644;R-332

【参考文献】