【作者】 魏东；

【导师】 刘英； 徐彤；

【作者基本信息】 甘肃农业大学 ， 基础兽医学， 2013， 博士

【摘要】 急性肺损伤（Acute lung injury，ALI）是由心源性以外的各种肺内外致病因素导致的急性、进行性加重的呼吸困难、难治性低氧血症和肺水肿，病情严重时发展为急性呼吸窘迫综合征（Acute respiratory distress syndrome，ARDS），重症者出现死亡。ALI是流感病毒感染哺乳动物的主要症状之一，其确切发病机理目前仍不很清楚，近期的研究表明，流感病毒引起的ALI可导致多器官的损害，近年来对其发病机理的研究主要集中在细胞因子和炎症介质的作用上。p38MAPK与炎症反应的调控密切相关，离体研究显示其参与调节多种炎性因子的表达。有资料显示，p38MAPK在肺损伤过程中发挥了重要的调控作用。研究发现，流感的历次大流行都与猪有关，猪流感病毒在人类流感的流行与病毒变异过程中较禽流感病毒具有更密切的关系。但有关p38MAPK在H9N2亚型猪流感病毒（H9N2-SIV）感染小鼠引起ALI的研究还末见相关报道。为了对p38MAPK在H9N2-SIV诱导小鼠ALI中作用进行研究，我们以6-8周龄SPF级BALB/c雌性小鼠为研究对象，利用本课题组分离的A/swine/HeBei/012/2008（H9N2）（H9N2-SIV），采用鸡胚尿囊腔接种法扩增病毒，经鼻腔滴鼻接种小鼠，以各组小鼠的临床症状、肺病理组织学变化、肺水肿的变化、支气管肺泡灌洗液（BALF）中炎性细胞的变化和血流动力学的变化等作为标准，建立H9N2-SIV诱导小鼠ALI模型，同时设立p38MAPK的特异性抑制剂SB203580处理组（SB组），选择小鼠ALI过程的不同时期进行测定，采用的主要方法：①观察小鼠的临床症状，统计小鼠死亡率，定时称量活鼠的体质量和采食量，测定小鼠肺系数、肺W/D、支气管肺泡灌洗液中炎性细胞的变化和血流动力学的变化；②建立H9N2-SIV感染小鼠的ALI模型；③应用HE染色技术、免疫组织化学技术和透射电镜技术动态观察肺脏的病理变化；④应用酶联免疫法测定炎症因子（TNF-α、IL-1β、IL-6、IL-10）的表达调控；⑤应用免疫组织化学技术和免疫印迹（Western-blot）技术测定磷酸化p38MAPK在肺组织中的分布及表达。测定结果显示：①感染组小鼠在感染病毒后2-3d开始发病，早期表现为背毛粗糙无光泽、活动减少、爱扎堆、精神沉郁、反应迟钝、食欲减退、眼睛缩小或紧闭、眼周围有分泌物；后期出现呼吸急促、被毛逆立、缩头弓背、拒食、消瘦等临床症状，蜷缩于鼠笼一隅，跑动缓慢或蹒跚样行走，个别小鼠出现明显的神经症状，死亡率达60%，体质量出现先降后升的趋势，小鼠出现严重肺水肿。从第8d开始症状逐渐减轻，14d后基本恢复正常。与对照组比较，感染组小鼠动脉血中氧分压从第2d开始降低，第4d出现明显的差异（P＜0.01），第6d最低时仅为（6.79±1.27）kPa，呈现严重的低氧血症，同时，二氧化碳分压显著上升（P＜0.05）；BALF内炎性细胞在感染后第4-8d显著增加，尤其以肺泡巨噬细胞和多核型白细胞增加最为明显（P＜0.01）。SB组小鼠体征表现比感染组减轻，比对照组加重，介于感染组和对照组之间。②病理组织学观察，感染组小鼠肺泡腔内有以嗜中性细胞为主的炎性细胞、红细胞和大量的粉红色的蛋白渗出物和水肿液，肺泡腔变小，肺泡壁增厚，肺泡间质不同程度增宽，细支气管和小血管周围间质水肿、疏松，可见中等量淋巴细胞和单核细胞浸润，支气管部分黏膜上皮细胞坏死，脱落；SB组血管及支气管壁周围稍有增厚、水肿，肺泡大小形态稍不规则，损伤程度亦明显小于病毒组。③感染组和SB组在各时间点肺组织匀浆中的TNF-α、IL-1β、IL-6和IL-10浓度均显著高于对照组（P<0.05）。感染组的TNF-α和IL-1β在损伤后第2d急剧升高，第4d达到高峰后开始下降，至14d后趋于正常；IL-6及IL-10于损伤后第2d开始升高，至第6d达到高峰，第8d虽然有所下降，但下降幅度不大，到14d仍高于正常对照组；SB组各时间点细胞因子水平较感染组降低，但仍高于对照组。④免疫组织化学测定，对照组小鼠肺组织基本正常，仅仅在肺泡隔上皮细胞和气道上皮细胞有极少量的磷酸化p38MAPK阳性表达。感染组磷酸化p38MAPK阳性表达增多，特别是肺泡隔中的上皮细胞几乎全部呈阳性，肺泡管结节状膨大部的肌样细胞同样呈阳性反应。同时磷酸化p38MAPK阳性表达还广泛分布于气道上皮细胞、血管内皮细胞、浸润炎性细胞的胞浆和胞核中，在浸润炎性细胞中表达量也较高；SB组相对于感染组磷酸化p38MAPK阳性表达明显减少，仅见肺泡隔中上皮细胞的胞浆有少量阳性表达，可见肺泡隔内的毛细血管内皮细胞阳性表达消失，至14d磷酸化p38MAPK阳性表达量基本恢复至对照组。⑤经Western-blot法检测，在H9N2-SIV感染小鼠的不同时间点，对照组小鼠肺脏有浅淡的磷酸化p38MAPK蛋白条带表达，但在对照组中任何观察时间点都未发现有统计学意义的变化。感染组小鼠磷酸化p38MAPK蛋白在感染后第2d，表达即明显增强，和对照组比较，差异有显著性（P<0.05）；至第6d蛋白表达达高峰，差异有极显著性（P<0.01），第8d磷酸化p38MAPK蛋白表达有所下降；到14d仍高于对照组。SB组小鼠肺脏在各时间点磷酸化p38MAPK蛋白表达均明显低于感染组，但仍高于对照组。通过对H9N2-SIV诱导小鼠ALI的研究，我们得出如下结论：本课题组分离的A/swine/HeBei/012/2008（H9N2）能够感染BALB/c小鼠，肺脏是其侵害的主要靶器官，引起小鼠的ALI，BALB/c小鼠可以作为流感病毒感染哺乳动物相关研究的良好模型动物；在ALI的病程中TNF-α、IL-1β起致炎因子的作用；IL-6在本试验中可能发挥抗炎作用，IL-10起抗炎因子的作用并在ALI的转归过程中发挥重要作用；p38MAPK能够被H9N2-SIV激活并介导ALI，p38MAPK参与了H9N2-SIV诱导小鼠ALI的发生和发展过程；p38MAPK特异性抑制剂SB203580可以下调磷酸化p38MAPK的表达水平，降低炎症因子的表达，减轻ALI中肺组织的病理损伤。更多还原

【Abstract】 Acute lung injury(ALI) refers to acute and progressively increased dyspnea, hypoxemiaand pulmonary edema caused by pathogenetic factors inside and outside the lung excludingcardiogenic ones. As the disease worsens, ALI develops into Acute respiratory distresssyndrome(ARDS), and even can be fatal to severe patients. ALI is one of the main symptomsof mammals infected by influenza virus. Its precise pathogenesis still remain unknown now.But recently, the research shows that AlI caused by influenza virus results in damages of manyorgans. These recent reseaches of pathogenesis focus on the function of cytokines andinflammatory mediator. p38MAPK is closely related to the regulation of the inflammatoryresponse. And in vitro studies shows that it is involved in regulating the expression of a varietyof inflammatory cytokines. According to data, p38MAPK plays an important regulatory role inthe process of lung injury. It is found that all previous pandemic influenza is associated withpigs, and the swine influenza virus(SIV) has a closer relationship in the process of humaninfluenza pandemic and virus variation than the avian influenza virus(AIV). But these relatedstudy that p38MAPK can cause ALI of mice infected by H9N2-SIV has not been reported.To study the role of p38MAPK on ALI of mice infected H9N2-SIV, We take6to8weeksof SPF BALB/c female mice as the research objects, use A/swine/HeBei/012/2008(H9N2)(H9N2-SIV) separated by this research group, and adopt the method of chicken embryoallantoic cavity inoculation to multiply the virus. And mice are infected through intranasalinoculation. The standard depends on change of clinical symptoms, histopathological changesin lung disease, pulmonary edema, the inflammatory cells in bronchoalveolar lavagefluid(BALF), and hemodynamics of each group. A mouse model of ALI induced byH9N2-SIV is built, and p38MAPK specific inhibitor——SB203580treatment group is alsobuilt. The different periods in the process of ALI of mice are measured. The principle methodsare as follows:①Observe clinical symptoms and count the mortality of mice, weigh body weight and feed intake of living mice regularly, determination of lung coefficient, W/D,the inflammatorycells in BALF, and hemodynamics of each group;②Bulid mice model ofALI induced by H9N2-SIV;③Dynamically observe the pathological changes of lung by the HE staining technique,immunohistochemical technique and transmission electron microscopy technology;④Measure the expression and regulation of inflammatory factor(TNF-α、IL-1β、IL-6、IL-10)by ELISA;⑤Measure the distribution and expression level of phospho-p38MAPK in lung tissue byusing immunohistochemistry and Western-blot.The results showed:①The mice of infected group got ill on2or3days post-infection (p.i.). The earlysymptoms were that back fur gets rough and unpolished, activities were less, and they loved toflock together; that they were depressed in spirit and slow in response; that they decreased inappetite, and the eyes were constricted or even closed, and covered with secretion. Then atlater stage, they appeared shortness of breath, reverse stand of back fur, retraction of heads andbend of backs, loss of appetite, and thinness etc.. Additionally, they crouched in a corner in thecage, and ran slowly or stumbled. And even some mice appeared obviously neurologicsymptoms. The death rate reached to60%. Body weight descended firstly then ascended, andmice were attacked by severe pulmonary edema. From8day p.i., the symptoms graduallyreduce, and basically recovered on14day p.i..On2day p.i.,the partial pressure of arterial oxygen(PaO2) for infected mice began toreduce compared with that of control group,which showed significant difference(P<0.01) on4day p.i.,and only6.79kPa±1.27kPa on6day p.i.,infected group appeared serioushypoxemia,moreover, the partial pressure of arterial carbon dioxide(PaCO2) rosesignificantly(P＜0.05).Inflammatory cells in BALF increased significantly,especially thealveolar macrophage and polymorphonuclear leukocytosis,and from4-8days after infectionshowed extremely significant difference(P<0.01) compared with that of control group.Physical symptoms of SB group were lighter than infection group, but severer thancontrol group, between infected and control group.②By the observation of the pathological histology, there were inflammatory cells mainlycontaining neutrophils, erythrocyte, a great deal of pink fibrinous exudate and edema fluid in the pulmonary alveoli of infected mice. And alveolar space became less, alveolar wall wasthickened, alveolar interstitial was widened to varying degree, Around bronchioles and smallvessels showed interstitial edema and loosen, moderate levels of lymphocytes and monocytewere infiltrated can be seen, and part of epithelial cells of bronchial mucosa underwentnecrosis and fall off; blood vessels and bronchus wall of SB group were slightly thickeningand edema, alveolar size were slightly irregular, and injury degree was also less thaninfected group.③Compared with the control group at each time point, the concentration of TNF-α、IL-1β、IL-6and IL-10in Lung tissue homogenate increased significantly in infection groupand SB group (P<0.05). TNF-α and IL-1β of infection group were remarkably increased on2day p.i., reached maximal levels on4day p.i. and then decreased, and finally almost returnedto the control levels on14day p.i.. IL-6and IL-10increased on2day p.i., reached maximallevels on6day p.i., then decreased on8day p.i., but drop slightly, and remained higher thancontrol group on14day p.i.. Cytokine levels of SB group decreased compared with infectiongroup at each time point, but were still higher than control group.④By the determination of immunohistochemistry, mice lungs in control group werenormal, but Positive expression of phospho-p38MAPK exsited in epithelial cell of alveolarsepta and airway in small quantities. Positive expression of phospho-p38MAPK in infectiongroup increased obviously, especially almost all of alveolar septa epithelial cells were positive,and myoid cells of the alveolar ducts nodular enlargement were positive too. Meanwhile,Positive expression of phospho-p38MAPK also exsited in cytoplasm and karyon of airwayepithelial cells, vascular endothelial cells, infiltrating inflammatory cells, and express quantityis higher in infiltrating inflammatory cells. The positive expression in SB group droppedsignificantly compared with infection group. It can only be founded in the cytoplasm ofAlveolar septa epithelial cells in small quantities. So positive expression of capillaryendothelial cells in alveolar septa disappeared. Positive expression of phospho-p38MAPKrecovered to level of the control group on14day p.i..⑤By the determination of Western-blot, mice lungs of control group have lightphospho-p38MAPK protein bands at different time point when mice were infected byH9N2-SIV. However, no significant changes were found in control group at any time point.Compared with control group, the expression of phospho-p38MAPK protein in infection group was stronger with significant difference(P<0.05) on2day p.i.,；it reached to the top withextremely significant difference (P<0.01) on6day p.i.,，decreased on8day p.i., but remainedhigher than control group on14day p.i.. The expression of phospho-p38MAPK protein in SBgroup was lower than infection group, but higher than control group.The following conclusions were drawn by this study. A/swine/HeBei/012/2008(H9N2)could infect BALB/c mouse. The lung is the main target organs, and it can cause ALI ofmouse. BALB/c mouse can be used as good animal model to study mammal infected byinfluenza virus. TNF-α and IL-1β play the role of inflammatory factors in the process of ALI,IL-6may play a role of anti-inflammatory action in this study, and IL-10also has the role ofanti-inflammatory factors and play an important part in the sequelae process of ALI.p38MAPK can be activated by H9N2-SIV and cause ALI, and p38MAPK takes part in theoccurrence and development of ALI induced by H9N2-SIV. p38MAPK specificinhibitor——SB203580can lower the expression of phospho-p38MAPK, and decrease theexpression of inflammation factors, lighten pathological injury of lung in ALI.更多还原