【作者】 胡晓红；

【导师】 段蕴铀；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2010， 博士

【摘要】 胸部开放伤后海水浸泡致ALI发病机制和早期干预的实验研究目的：现代海战中,胸部开放伤后海水浸泡占海战伤落水人员死因的第2位。海水具有高渗、高钠、低温等特点,胸部开放伤后海水浸泡致急性肺损伤(SI-ALI)既有创伤因素,又有海水浸泡因素,发病机制错综复杂,目前尚未完全阐明,亦缺乏针对发病机制实施的有效救治措施。国内外关于海战伤的报道非常罕见,国外未见文献报道,国内上世纪九十年代末期和本世纪初期海战伤的研究重点在战伤(胸伤、腹伤、四肢伤、颅脑伤、烧伤及休克等)合并海水浸泡的伤情特点及早期外科救治,对于海战伤后重要脏器衰竭发生、发展的病理机制及救治和预后未进行系统研究。而进一步研究海战伤后重要脏器衰竭的发生机制,制定针对性的救治措施,是实施快速有效救治,提高生存率不可缺少的环节,对维护和增强参战部队官兵战斗力具有十分重要的意义。因此本研究目的是探讨海水高渗因素、血管内皮生长因子(VEGF)和前B细胞集落促进因子(PBEF)等致病因子在胸部开放伤后海水浸泡致急性肺损伤早期发生中的作用以及地塞米松早期干预的意义。内容：复制薛志强等改良的SI-ALI犬模型；通过比较胸部开放伤后海水浸泡与淡水浸泡致犬肺组织损伤的伤情差异,探讨海水高渗透压这一特性在SI-ALI早期发病中的重要作用；通过观察VEGF及其受体VEGFR-2、可溶性受体sVEGFR-1在SI-ALI犬模型早期血浆和肺组织内水平变化,探讨VEGF作为一个重要的血管通透因子在SI-ALI早期肺水肿发生过程中的作用；通过观察SI-ALI早期PBEF在血浆和肺组织内水平变化,探讨PBEF与胸部开放伤后海水浸泡致ALI早期炎症反应的关系；通过观察早期地塞米松治疗后SI-ALI犬模型血浆炎症因子和VEGF水平的变化,探讨早期地塞米松干预对胸部开放伤后海水浸泡致ALI的保护作用。方法：将32只实验用犬随机均分为对照组(CG,单纯胸部开放伤组),淡水组(FG,胸部开放伤后胸腔淡水浸泡组)、海水组(SG,胸部开放伤后胸腔海水浸泡组)、以及地塞米松组(DG,海水浸泡后早期地塞米松干预组),共4组进行实验研究。麻醉后经口腔气管插管,用锐器于实验犬右胸第4肋间制成直径0.5cm切口,放置一内径0.3 cm塑料导管使胸膜腔与大气相通,形成开放性气胸：胸壁伤口开放5 min后缝合封闭伤口作为对照组；切口与大气相通5 min后立即用50 ml注射器经胸壁切口向胸腔内缓慢注入淡水(35 ml/Kg),作为淡水组；经伤口灌入海水入胸腔(35ml/Kg),作为海水组；5 min灌注结束,并缝合伤口使之封闭；在海水灌注30 min后经颈静脉注射地塞米松(1 mg/kg),作为地塞米松组。分别于胸部开放伤前(0 h)、胸部开放伤后2、4、6和8 h留取血样本,8 h取支气管肺泡灌洗液(BALF)样本和肺组织样本。肉眼观察肺组织大体病变,HE染色镜下观察肺组织病理改变；常规实验室方法检测电解质浓度和血浆渗透压(POP)变化,测血浆和BALF总蛋白含量并计算肺泡通透指数(PVPI); ELISA方法测血浆炎症因子(IL-1β、IL-8、vWf等)、VEGF及受体和PBEF水平；免疫组织化学方法(IHC)观察肺组织VEGF、PBEF分布,Western印迹法测肺组织VEGF和PBEF蛋白表达,Real-time RT-PCR方法检测肺组织中编码VEGF和PBEF的mRNA合成。用SPSS 13.0统计软件包处理数据,P<0.05为差异有显著意义。结果：1.模型复制成功：海水浸泡组犬均表现为呼吸急促、进行性呼吸困难,PaO2显著下降,伤后6 h氧合指数(PaO2/FiO2)均<300 mmHg(40Kp),符合急性肺损伤(ALI)的诊断标准；组织学改变也符合ALI病理改变,HE染色光镜下见：肺泡和肺间质内广泛充血、水肿液积聚,肺泡间壁明显增宽,大量白细胞渗出、聚集,部分区域肺泡结构的完整性破坏,肺泡融合,部分区域肺泡塌陷萎缩、含气减少,毛细血管扩张淤血,胸部开放伤后海水浸泡致ALI犬模型复制成功。对照组和淡水组伤后出现呼吸急促、PaO2下降,伤后2 h后逐渐缓解,氧合指数始终>300 mmHg(40Kp),未达到ALI诊断标准。2.淡水组和海水组伤情比较：①与淡水组比较,海水组肺泡通透指数(PVPI)、血浆渗透压(POP)、炎症因子IL-1β、IL-8和vWf水平明显升高,与淡水组有显著差异(P<0.05)；②淡水组炎症因子IL-1β和IL-8较对照组显著升高,POP降低,与对照组有显著差异(P<0.05)；③淡水组PVPI和vWf水平与对照组无统计学差异(P>0.05)；④相关分析显示POP与炎症因子IL-1β、IL-8,与PVPI明显正相关(P<0.05)。3.胸部开放伤后海水浸泡致ALI早期VEGF水平变化：与对照组比较,①海水组血浆VEGF及其受体明显增加,肺组织中的VEGF蛋白及编码VEGF蛋白的mRNA亦明显增加,较对照组有显著差异(P<0.05)；对照组内血浆、肺组织中VEGF及其受体水平、VEGF mRNA与伤前无显著差异；②海水组外周血白细胞、IL-1β、IL-8、vWf较对照组显著升高(P<0.05)；③海水组IL-1β和IL-8在伤后2-4h较伤前明显升高,而对照组内白细胞、IL-1β和IL-8在伤后6-8 h较伤前升高,海水组较对照组升高明显提前,且有显著差异(P<0.05)；对照组vWf与伤前无显著差异(P>0.05);④VEGF水平变化与炎症因子的升高相一致；相关分析显示VEGF水平与血浆渗透压(POP)、肺泡通透指数(PVPI)明显正相关。4.胸部开放伤后海水浸泡致ALI早期PBEF水平变化：与对照组比较,①海水组血浆PBEF明显增加,肺组织中PBEF蛋白及PBEF蛋白编码mRNA亦明显增加,较对照组有显著差异(P<0.05)；②对照组血浆和肺组织中PBEF较伤前明显增加,但与海水组仍有显著差异；③PBEF升高与炎症因子IL-1β和IL-8明显正相关。5.早期地塞米松干预：与海水组比较,①早期地塞米松治疗组外周血白细胞、IL-1β、IL-8、vWf水平在治疗后6h显著减低,肺泡通透指数(PVPI)亦显著减低(P<0.05)；但与对照组伤后同时间点相比,仍有显著差异；②治疗后血浆渗透压(POP)、电解质浓度与海水组差异无统计学意义(P>0.05)；③早期地塞米松治疗组血浆VEGF及其受体水平升高的峰值显著降低、升高进程延迟,但仍明显高于对照组。结论：1.海水加重全身炎症反应和肺毛细血管内皮细胞损伤,是胸部开放伤后胸腔海水浸泡致急性肺损伤的重要因素。2.胸部开放伤后海水浸泡致ALI发生早期,血浆、肺组织中的VEGF水平均明显升高,导致肺泡毛细血管膜通透性增高。3.胸部开放伤后海水浸泡致ALI早期PBEF水平升高,参与炎症反应的发生发展,致肺泡毛细血管膜屏障损伤,促进急性肺损伤的形成。4.早期小剂量地塞米松干预可减轻炎症反应,降低VEGF水平、改善肺微血管通透性,对胸部开放伤后胸腔海水浸泡致ALI具有一定的保护作用。更多还原

【Abstract】 ObjectiveIn the modern sea conflict, soldiers with open chest trauma were very susceptible to fall into the sea, and immersed in seawater. Subsequently, acute lung injury (ALI) would happen following open chest trauma and seawater-immersion of the thoracic cavity. Acute lung injury induced by seawater-immersion following open chest trauma (SI-ALI), which further developed to acute respiratory distress syndrome (ARDS) and the multiple organ dysfunction syndrome (MODS), is associated with high mortality. Unfortunately, the pathogenesis and management of these complex and often lethal syndromes have been unclear. Though the lung would be injuried heavily and resulted in severe and intricate physiopathologic changes, there is rare report from overseas and a few from domestic about this special ALI. Li Hui and colleagues reported the clinical manifestations and drew up some measures to the trauma on the early onset of SI-ALI. Professor Duan and our team expended considerable effort to study the comprehensive mechanisms and treatment measures of acute lung injury following open chest trauma and seawater-immersion.Pathogenic factors, such as seawater, cytokines, may play crucial role in the pethogenesis of SI-ALI. Higher osmotic pressure, lower temperature and abundance of salts are the mainly characteristics of seawater. On the physiological action, the role of VEGF on endothelial cell permeability and proliferation, have been demonstrated in studies. But there are no simple answers to the pathological issue, such as ALI, which require an understanding on the roles not only VEGF lead to acute pulmonary edema, but also VEGF protects the epithelial cells from cell death and activate endothelial cell proliferation. Pre-B cell colony-enhancing factor (PBEF), highly conserved in evolution, is a 52-kDa protein found in living species from bacteria to humans and is an endogenous inflammatory mediator. Recently, PBEF was discussed as a novel biomarker for ALI because of its inhibitting apoptosis of neutrophils in ALI. Despite extensive research and increasing awareness of ALI, few studies have been done on the relation between PBEF and ALI induced by seawater-immersion after trauma. We knew a little about their roles in SI-ALI. Therefore, in this study, we would focus on discussing the role of these pathogenic factors in the pathogenesis of SI-ALI. Furthermore, we want to give evidence that dexamethasone attenuate the inflammatory response, at least partially. Today’s aim is, firstly, to investigate the pathophysiological changes through comparing the differences of the injury respectively by seawater-immersion or freshwater-immersion following open chest trauma in dog models; secondly, to investigate the levels of VEGF and PBEF in SI-ALI and further explore whether the changes of VEGFs and PBEF are connected with the pathogenesis of SI-ALI. Thirdly, to evaluate whether the low-dose dexamethasone treatment on the early onset of SI-ALI could act as an inhibitor of systemic inflammation and prevent or attenuate the lung injury following open chest trauma and seawater-immersion in dog models.MethodsAll investigations involving experimental dogs were reviewed and approved by the Institutional Review Board (IRB) of Beijing Navy General Hospital Animal Care and Use Committee. We prefer large animals for their essential tolerance to fulfill the hard experimental process. Dogs were randomly divided into four groups:control group (CG), seawater group (SG), freshwater group (FG) and dexamethasone treatment group (DG). All dogs were anesthetized with intramuscular injection of ketamine (20mg/kg) and assisted ventilation with endotracheally intubated. A 0.5 cm diameter incision was made with a sharp instrument between the 4 th and 5 th ribs and into the right chest and a hollow plastic tube was placed into the incision for 5 min and then pull it out to form an opened pneumothorax. The dogs in CG only suffered from pneumothorax, whereas seawater or freshwater (35ml/kg) was slowly infused into the pleural cavity of animals in SG or FG, respectively. The incision skins of all experimental dogs were sutured 10 min after trauma. The right carotid artery and jugular vein were cannulated for drawing blood. Fluid or air in the chest made it difficult to breathe. Arterial blood gas values were monitored and the oxygen index (PaO2/FiO2)<300 mmHg is the standard to prove the success of the ALI model and all experimental dogs met the criteria for ALI.Blood samples were collected at 0 h before trauma and 2,4,6 and 8 h after trauma. At 8 h, lungs were lavaged thrice with 15 ml of saline through the endotracheal tube and gently aspirating back to collect the broncho-alveolar lavage fluids(BALF).Whereafter, the dogs received an intracardiac injection of 15 ml of 15% KCl and were then sacrificed. The lungs were removed for histological evaluation. The levels of total protein both in plasma and BALF were measured to calculate the pulmonary vascular permeability index (PVPI). Lung injury was assessed by histology. The levels of VEGF, PBEF and inflammatory cytokines in plasma were measured by ELISA Kit. The levels of PBEF and VEGF protein in lung tissue were evaluated with immunohistochemistry and Western blotting. Quantification both of VEGF mRNA and PBEF mRNA were evaluated in lung tissue by real-time reverse transcriptase-PCR. Data were compared between groups using ANOVA analysis.Results1. The SI-ALI dog model copied successfully. All animals in "SG" represented increasing breath frequency and breath distress following trauma. Arterial oxygen partial pressure (PaO2) decreased markedly. Arterial oxygen partial pressure/suction gas oxygen concentration (PaO2/FiO2) which oxygenation index dramatically reduced in 2 h and continuely reduced in 6-8 h after trauma, and maintained less than 300 mmHg, which met the criteria of ALI. The pathological changes in the "SG" demonstrated a marked lung injury, represented by hemorrhage, edema, thickened alveolar septum, formation of hyaline membranes and the infiltration of inflammatory cells in alveolar spaces.2. Though arterial oxygen partial pressure (PaO2) and arterial oxygen partial pressure/suction gas oxygen concentration (PaO2/FiO2) decreased in "FG", they didn’t meet the criteria of ALI. Compared with the samples from "FG", plasma osmotic pressure (POP, mmol/L) in "SG "was significantly increased (SG vs. FG: 326.66±3.45 vs.268.61±4.40, P<0.05); the pulmonary vascular permeability index (PVPI) significantly increased in "SG "(SG vs. FG:0.055±0.002 vs.0.034±0.007, P <0.05); the cytokines levels (pg/ml) were markedly increased in "SG" (IL-1β: 123.82±46.39 vs.66.53±13.88; IL-8:79.91±8.24 vs.57.69±8.37; vWf:1.03±0.09 vs. 0.64±0.08, P<0.05); the levels of PVPI, IL-1βand IL-8 were significantly positively correlated with the plasma osmotic pressure (rho:0.743,0.616 and 0.638, P<0.05).3. Compared with animals in "CG", the levels of both IL-1βand IL-8, POP, PVPI significantly increased in "SG", P<0.05; The plasma levels of both VEGF and sVEGFR-1 (pg/ml) significantly increased in "SG" (SG vs. CG, VEGF:72.20±25.41 vs.27.28±2.27, P<0.05; sVEGFR-1:1.92±0.32 vs.0.61±0.13, P<0.05); The level of VEGF protein in lung tissue was increased in "SG", (SG vs. CG,0.2375±0.036 vs.0.1649±0.031, P<0.05); The level of VEGF mRNA synthesis in lung tissue was increased in "SG", (SG vs. CG, VEGF mRNA:5.04±0.29 vs.0.25±0.04, VEGFR-2 mRNA:5.08±0.20 vs.0.64±0.02, P<0.05). The levels of VEGF was significant correlatiom with POP and PVPI.4. Compared with animals in "CG", PBEF levels (pg/ml) significantly increased in "SG" (plasma:3014.16±883.47 vs.1060.94±251.08, P<0.05; BALF:1373.35±102.53 vs.997.77±70.31, P<0.05; lung tissue:1455.22±71.74 vs.921.43±118.13, P<0.05); PBEF level were positively correlated with IL-1βand IL-8 (IL-1β: rho is 0.820, P<0.05; IL-8:rho is 0.842,P<0.05).5. Compared with the samples in "SG", the levels of IL-1βand IL-8 from plasma (pg/ ml)were markedly decreased in "DG". (IL-1β:90.97±41.99 vs.144.11±29.72; IL-8: 45.21±16.39 vs.88.26±6.66, P<0.05); In lung tissue, the levels of IL-1β(40.18±10.59 vs.60.72±10.87, P<0.05) and IL-8 (34.63±5.55 vs.49.39±8.61,P <0.05) were decreased significantly in 8 h after treatment. The PVPI was markedly decreased too (0.039±0.006 vs.0.055±0.002, P<0.05); But there was no significant difference of electrolyte concentration and POP between these two groups (P>0.05).Conclusions1. The injury of immersion in seawater following open chest trauma is severer than that of in freshwater. It is attributed to the seawater’s characteristic of higher osmotic pressure and abundance of salts, which increasing the PVPI and aggravating the inflammatory response.2. Higher levels of VEGF and it’s receptor in plasma and lung tissue are distinguishing characteristics during the early onset of SI-ALI. Furthermore, the levels of VEGF in the early onset of SI-ALI were significantly positive correlated with POP and PVPI. Early release of VEGFs increase pulmonary vascular permeability and partially lead to the development of SI-ALI. VEGFs may have a crucial role in the early onset of SI-ALI.3. Higher levels of PBEF in the early onset of SI-ALI were significantly positive correlated with inflammatory factors. As a proinflammmatory and destructive mediator, PBEF have important roles in the development of SI-ALI.4. low-dose dexamethasone have an protection on the early onset of SI-ALI through decreasing the inflammatory response and improving the pulmonary permeability.更多还原