【作者】 杨晓文；

【导师】 金发光；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 硕士

【摘要】 目的:旨在以油酸造成SD大鼠急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）动物模型为研究对象,通过血气分析、观察肺组织病理、肺泡Ⅱ型上皮细胞形态改变来评价早期肺组织损伤的程度,测定AQP4在肺组织及肺泡Ⅱ型上皮细胞表达,进而观察特布他林气管滴入治疗时,肺组织病理改变、血管外肺水（EVLW）的变化以及对肺组织及肺泡Ⅱ型细胞AQP-4的表达影响,从而分析特布他林药物在急性肺损伤时如何通过影响肺水肿形成和上皮细胞来清除液体量变化,进而影响肺水肿的形成,为研究刺激β₂受体cAMP途径对水通道蛋白的影响和可能存在的机制提供理论依据。方法:1.建立油酸肺损伤大鼠模型选用2月龄二级雄性SD（Sprague Dawley）大鼠60只,实验室适应性喂养1周后,体重220±20克。按照序号完全随机分为正常对照组、油酸制ALI肺损伤模型组,特布他林气道治疗组。每组各20只大鼠。三组大鼠均行气管插管,模型组和特布他林组经腹腔静脉注入油酸0.1ml/kg后,对照组注入等量生理盐水。15分钟后特布他林组气管插管内滴入特布他林2.5mg/kg,余各组滴入等量的生理盐水。30分钟后抽腹主动脉动脉血0.3ml血气分析判断肺损伤的程度。2.致伤组大鼠肺组织损伤评价腹腔静脉注射油酸0.1ml/kg,30分钟后抽血气分析;治疗后30分钟腹主动脉放血活杀。通过HE病理染色采用Smith评分法对肺水肿、肺泡及间质炎症、肺泡及间质出血、肺不张和透明膜形成五项分别进行0～4分标准半定量分析[1]:0级为无该项病理改变;1级为病理变化轻且很局限;2级为病理变化中等且局限;3级为病变中等但广泛或局部很显著;4级为非常显著的广泛性病理改变,求出各动物肺的各项病理改变程度的平均程度,作为肺损伤分数。动脉血气分析判断大鼠肺损伤模型是否成功建立。3.大鼠ATⅡ细胞的分离、纯化和鉴定模型组、特布他林组及正常对照组的大鼠,取其肺组织,做好标记,分别采用改良的胰酶消化法分离肺泡Ⅱ型上皮细胞,用大鼠IgG免疫粘附法纯化细胞,台盼兰测细胞活力,改良的鞣酸染色法和透射电镜鉴定细胞纯度。4.AQP-4大鼠肺泡组织及肺泡Ⅱ型上皮细胞m-RNA表达的测定免疫组织化学试剂盒进行肺组织AQP-4免疫组化染色;Trizol提取肺泡Ⅱ型上皮细胞总RNA,紫外线分光光度仪OD260检测其含量。逆转录-聚合酶链反应（RT-PCR）检测AQP-4mRNA的表达。采用Strom 860图像分析系统行密度扫描,计算AQP4产物与β-actin产物吸光度积分的比例,作为AQP4mRNA表达水平。5.血管外肺水（EVLW）的测定重力法测定EVLW。放血活杀切取完整肺组织,去除支气管,通过检测分布于肺血管外的液体,主要为细胞内液、肺泡内液和肺间质液变化反映肺水肿的程度。根据公式计算EVLW含量。结果:1.大鼠造模后的评价油酸注入后大鼠均在30s-3min内出现明显呼吸加快,浅促,口唇皮肤以及四肢发绀,气管插管内分泌物明显增多的临床表现。血气分析PaCO2明显上升,PO2下降,PaO₂/FiO₂值符合ALI的诊断标准。HE染色病理示肺间质及肺泡内有小量出血,可见炎性细胞增多;肺泡隔增宽,肺间质渗液明显。2.大鼠ATⅡ细胞的分离纯化方法的评价经台盼兰染色对照组细胞活力84.3±3.21%,油酸组83.7±2.96%（图10-11）,电镜下见细胞形态较规则,呈圆形或立方形,表面有很多长短不一的微绒毛,核圆形,胞质着色浅,见特征性的嗜锇性板层小体（图12）。ALI组电镜下见细胞变性,细胞形态相对不规则,表面的微绒毛明显减少,胞核变形,染色质边集,线粒体肿胀,胞浆内各种细胞器明显减少,板层小体排空显著增多而致空泡化。3.AQP4肺泡组织免疫组化染色及肺泡Ⅱ型上皮细胞的表达正常组AQP4呈弱阳性染色,细胞偶被染成淡黄色（图）,主要分布肺泡上皮细胞形态较规则的细胞上,在ALI组AQP4呈阳性染色,棕黄阳性颗粒增多、染色强度变大。肺泡Ⅱ型细胞提取RNA经过RT-PCR扩增后,ALI组合成明显增加。4.特布他林治疗后血气分析、病理及血管外肺水（EVLW）含量变化特布他林组血气分析未达到ALI诊断的标准,PO2增高;病理Smith评分较模型组明显下降,具有统计学意义;EVLW明显小于模型组,明显提高肺泡上皮液体清除。5.特布他林对肺泡及肺泡Ⅱ型上皮细胞AQP-4影响特布他林组AQP4免疫组化呈强阳性表现,主要分布肺泡上皮细胞形态较规则的细胞上（图29、30）。肺泡Ⅱ型上皮细胞扩增与对照组有显著性差异,吸光度积分较模型组增加,但二者未有显著性差异。结论:模型组血气分析,肺部病理改变,上皮细胞的超微结构及EVLW严重程度显著高于对照组,符合肺损伤及急性呼吸窘迫综合征的诊断标准,说明腹腔静脉注射油酸建立大鼠ALI急性模型是一个相对简单而成功率高的方法。采用改良的胰酶消化法可以成功分离正常和油酸ALI大鼠的ATⅡ细胞,用大鼠IgG包被培养皿纯化细胞、鞣酸染色法鉴定细胞纯度简单可行,而透射电镜是鉴定细胞的金标准。显微镜下肺泡Ⅱ型上皮细胞的数量较对照组无明显减少,但电镜下细胞的超微结构如板层小体排空,线粒体损伤表现。通过免疫组化证明AQP4存在于大鼠肺泡上皮,尤其Ⅱ型细胞膜。RT-PCR显示模型组ATⅡ细胞受损后,AQP4表达增高,说明其仍具有上调水通道蛋白转运功能。气道内特布他林给药,从肺脏大体、血气分析、病理、肺泡Ⅱ型上皮细胞电镜下形态等结果达不到肺损伤的标准,肺毛细血管渗透性下降,间质水肿减轻,肺泡上皮细胞主动重吸收加强,体现在血管外肺水明显下降,AQP4免疫组化强阳性,说明AQP4由胞浆向胞膜转移增多及RT-PCR证明细胞核合成AQP4增多,能够促进并发挥AQP4肺泡腔液重吸收作用,可能是早期肺损伤促进肺水肿的重吸收作用代偿机制之一。更多还原

【Abstract】 ObjectivesTo study an acute lung injury/acute respiratory distress syndrome（ALI/ ARDS）animal model by oleic acid in rats with stability and repeatability used to investigate lung tissue pathology,alveolar typeⅡepithelial cell morphology and water content of extravascular lung water（EVLW）Early changes in lung tissue to evaluate the extent of injury determined by AQP4 in lung tissue and alveolar typeⅡcells and then observe the tracheal instillation of terbutaline therapy,analysis of the pathological changes of lung tissue,EVLW determine changes in pulmonary edema formation and clearance liquid volume of poor lung tissue and alveolar typeⅡcells,AQP-4 expression differences,and thus analysis of terbutaline in acute lung injury is how it affects the mechanism of pulmonary edema to study theβ2 receptor to stimulate the cAMP pathway on the water channel protein impact and provide a theoretical basisMethods1.Establish the model of lung injury in rats by oleic acidThirty male Sprague-Dawley rats of clean grade were used to experiment after one week of adaptive feeding in our laboratory,weighing 220±20 g. According to the serial number,We divided 3 groups concluding control group、oleic acid induce ALI group、terbutaline therapy group.Three groups underwent tracheal intubation,model group and the terbutaline group of intravenous injection of oleic acid by intraperitoneal vein 0.1 ml/kg after the injection of normal saline control group.15 minutes after terbutaline instillation with endotracheal intubation group terbutaline 2.5mg/kg,more than the equivalent of normal saline instillation group.30 minutes after the abdominal aortic artery pumping 0.3ml blood gas analysis to determine the extent of lung injury.2.Assess the rats’ lung injury after injection of oleic acidIntravenous injection of oleic acid,30 minutes after the blood gas analysis; after 30 minutes the bloodletting through abdominal aorta.HE staining through the use of Smith pathological score on pulmonary edema,alveolar and interstitial inflammation,hemorrhage,atelectasis and hyaline membrane formation were analysis of from 0 to 4（11）,arterial blood gas analysis to determine lung injury in rats the extent of the establishment of the model.3.Isolating,purifying and identifying of alveolar typeⅡcells in ratsmarked lung tissue of choice,using trypsin digestion method improved the separation of typeⅡalveolar epithelial cells,using rat IgG purified cell immune adherence,Trypan blue cell viability test,improved tannin staining and transmission electron microscopy to identify cell purity.4.AQP-4 express in lung tissue and ATII cells of ratsKit immunohistochemistry for AQP-4 in lung tissue by immunohistochemical staining;Trizol extraction of alveolar typeⅡcells total RNA,UV Spectrophotometer content OD260 detected.Reverse transcription-polymerase chain reaction（RT-PCR）detected the expression of AQP-4mRNA.Strom 860 using image analysis system scanning line density to calculate the product of AQP4 andβ-actin product of the ratio of absorbance points,as the expression level of AQP4m-RNA.5.To test Extravascular lung water content（EVLW） EVLW were examined at 3 groups respectively after infusion by gravity. Lung tissue removed bronchial,by detecting the distribution of pulmonary blood vessels in the outer liquid,Liquid extrude alveolar space by hydrostatic pressure and osmotic pressure.EVLW contents in accordance with formula.Results1.Assessment of the oleic acid rat modelsAfter injection of oleic acid for more than 30s,most experimental animals appeared obviously symptoms such as short of breath,skin of face and legs became purple.PaO₂ and PH were significantly decreased and PaCO₂ was increased obviously apply to diagnostic criteria of ALI/ARDS.The pathological changes of lungs seem to be engorgement,edema,bleeding,etc.2.To assesse the methods of isolation and purification ATⅡcellsThe improved methods we used to isolate and purify ATⅡcells were feasible.Using these methods,we were able to obtain an ATⅡcell population with 84.3±3.21%confirmed by 4%Trypan blue solution,and confirmed by staining for tannic acid,we got 83.7±2.96%purity of oleic acid group.Decreasing the concentration of digestive enzyme and centrifugal rate could raise the cellular vitality.It could be found the lamellar bodies with a tannic acid stain and electron microscope.3.Expression of AQP4 in Alveolar organization and alveolar typeⅡcellsAQP4 was present at the membranes of isolated rat alveolus lung by immunohistochemical staining.The distribution of aquaporin-4（AQP-4）on the cellular membrane was observed and it also stained strongly in ALI rat alveolar cells.4.To analysis blood gas analysis,pathology,and EVLW after terbutaline treatmentAfter Terbutaline therapy blood gas analysis does not match the criteria for ALI diagnosis,PO2 increased;Smith pathology score than the model group decreased,with statistical significance;EVLW decreased significantly reflect that terbutaline may has a therapeutic effect in early lung injury,markedly improved alveolar epithelial fluid clearance.5.Terbutaline affect AQP-4 on alveolar and alveolar typeⅡepithelial cellsAQP4 in the therapy group is expressed mostly in three groups.It is demonsteated that AQP4 was up-regulated at the membranes of rat alveolar cells in injuried and therapy stage.Through RT-PCR of amplification,the terbutaline group is significantly increased,followed by the model,it could explain that AQP4 of m-RNA is increasing in the nucleus of ATⅡand terbutaline may stimulate the expression.ConclusionsIn this model,the rat Blood gas analysis,Smith lung injury score in ALI group was worser than those of control group under microscope and blood gas analysis.The practice indicated that setting up rat ALI model by injection of oleic acid through coeliac vena was an easy and efficient method.We had successfully isolated and purified ATⅡcells from normal and ARDS rats.It is simple and feasible that staining for tannic acid to identify purity of cells.Electron microscope is the best way to authenticate these cells.Alveolar typeⅡunder the microscope the number of epithelial cells than the control group is no significant decrease,cells have ultrastructure injury in the electron microscope.By immunohistochemistry to prove that AQP4 is present at the membranes of alveolar epithelial cells and isolated rat typeⅡcells.RT-PCR showed that the model group after ATⅡcell damage,AQP4 expression increased,its still on the water diversion channel protein with transport function.Terbutaline administration by air tube,the results which draw from observation of the lungs, blood gas analysis,pathology,alveolar typeⅡepithelial cells are less than the standard lung injury.When rats in ALI get therapy of terbutaline the expression of AQP4 in typeⅡcells is significantly increased as compared with control group.AQP4 was strongly positive immunohistochemical staining shows that AQP4 transfer from the cytoplasm to the membrane and an increase in cell RTPCR proved to increase synthesis of AQP4,AQP4 could play to promote and alveolar space fluid re-absorption may be the promotion of early lung injury pulmonary edema re-absorption of compensatory mechanisms.更多还原