重症急性胰腺炎肺损伤大鼠肺内血管生成素样蛋白4、细胞间黏附分子-1基因表达及意义

目的：探讨重症急性胰腺炎肺损伤大鼠肺内的血管生成素样蛋白4(ANGPTL4)、细胞间黏附分子-1(ICAM-1)基因表达及意义。方法河北大学实验动物中心提供48只体质量280~340 g 的健康成年雌性 SD 大鼠,对照组大鼠(8只)开腹后,只对十二指肠翻动后关腹;实验组大鼠(40只)每一时间段选用 8只,分别在制模之后1小时、3小时、5小时、7小时和9小时处死实验动物,再行相应检测,包括肺组织 ANGPTL4、ICAM-1,肺髓过氧化物酶(MPO)的活性,肺微血管的通透性,肺组织的含水量等。结果对照组肺组织内 ANGPTL4、ICAM-1基因表达呈低水平,制模1小时后开始上升,而后不断上升,至9小时达到峰值,各时段间比较差异均有统计学意义(P <0.05)。结论 ANGPTL4、ICAM-1的高表达与重症急性胰腺炎肺损伤存在着紧密的联系,也为重症急性胰腺炎肺损伤防治提供了一条新的方法和思路。     

PI3K/PKB信号转导通路活性在重症急性胰腺炎急性肺损伤中的变化

目的 探讨重症急性胰腺炎急性肺损伤中磷脂酰肌醇3-激酶/蛋白激酶B(P13K/PKB)信号转导通路的活性变化规律.方法 健康成年雄SD大鼠40只,随机分为SO组(n=10)、SAP组(n=30),BD针胰胆管逆行注入牛磺胆酸法建立重症急性胰腺炎急性肺损伤大鼠模型.SAP组3、6、12h检测血淀粉酶、血气分析、肺含水量,肺组织髓过氧化物酶(MPO)活性,光镜下进行胰腺和肺组织病变程度评分.免疫组化法检测肺组织ICAM-1和P-PKB的表达,蛋白印迹(Western blot)法检测PI3K/PKB信号通路的活性变化.SO组在术后12h检测上述相关指标.结果 肺损伤程度逐渐加重,组织含水量及MPO活性逐渐升高(P<0.05),PaO_2明显降低(P<0.05).制模后3h随p-PKB的活性逐步增强,ICAM-1表达逐渐增多,两者呈正相关(r=0.910,P<0.05);PI3K/PKB信号通路逐步被激活,至12h达到峰值,与肺损伤程度呈正相关(r=0.871,P<0.05).结论 P13K/PKB信号传导通路被激活是重症急性胰腺炎急性肺损伤的重要发病机制之一.     

特布他林影响水通道蛋白4在大鼠急性肺损伤肺水代谢的作用

目的 油酸造成急性肺损伤(ALI) 初期,特布他林气管滴入治疗,根据肺损伤程度及测定血管外肺水的大小,评估病理状态下特布他林对肺泡Ⅱ型上皮细胞(ATⅡ) 水通道蛋白4(AQP-4 )转运水能力的影响.方法 血气分析、肺组织病理学评分、透射电镜检测肺损伤程度;重力法测定血管外肺水(EVLW)含量;免疫组化法分析肺组织AQP-4的表达,急性分离纯化ATⅡ细胞,电子显微镜观察超微结构;逆转录-聚合酶链反应(RT-PCR) 检测肺泡Ⅱ型上皮细胞AQP-4的mRNA 表达.结果 血气分析、光镜下肺损伤评分说明油酸致大鼠急性肺损伤,显微镜下ATⅡ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,线粒体损伤等改变;特布他林治疗后血气分析、病理评分、电镜下细胞等损伤明显减轻(P<0.05);EVLW三组间有显著性差异(P<0.05),治疗组肺水下降明显;肺组织免疫组化观察到损伤后肺泡上皮细胞AQP-4表达增强,呈阳性,特布他林组表达呈强阳性;ATⅡ细胞AQP-4的mRNA 表达治疗组、油酸组较对照组有显著性差异(P<0.05),两者之间无显著性差异(P＞0.05).结论 特布他林可能通过上调肺泡上皮细胞和Ⅱ型细胞上AQP-4的表达,介导水转运减轻肺损伤时肺泡腔水肿.     

核因子-κB在小鼠急性肺损伤中的作用

目的　探讨核因子 (NF) κB在内毒素 (LPS)诱导的急性肺损伤小鼠发病中的作用。方法　腹腔内注射LPS诱导小鼠急性肺损伤模型。LPS注射后 0、 1、 3、 6、 12测定肺湿重 /干重比值 (W/D) ,迁移率改变电泳法检测肺组织NF κB活性 ,同时酶联免疫吸附法测定肺组织匀浆中肿瘤坏死因子 (TNF) α、白介素 (IL) 10浓度 ,RT PCR检测mRNA表达。结果　LPS注射后 ,W/D比值明显增高 ,6h升高最明显 (4 82± 0 10 ) ,显著高于LPS注射前 (3 6 7± 1 0 4 ,P <0 0 5 )。LPS注射后肺组织核蛋白NF κB活性明显增强 ,6h达到峰值 (40 5 7± 6 2 4 ) ,显著高于LPS注射前 (44 8± 30 9,P <0 0 5 )。肺组织匀浆TNF α和IL 10浓度分别在LPS注射后 6h和 12h升高最明显 ,分别为 (197 1± 5 2 4 )pg/ml和 (6 4 9± 39 7)pg/ml,显著高于LPS注射前 [分别为 (6 1 2± 10 7)pg/ml和 (71 6± 15 9)pg/ml]。与LPS注射前比较 ,LPS注射后 3～ 12h ,肺组织匀浆TNF α和IL 10mRNA表达显著增高。肺组织病理显示肺泡出血、水肿、大量炎症细胞浸润 ,电镜下见Ⅰ型肺泡上皮细胞断裂 ,Ⅱ型肺泡上皮细胞变性。结论　LPS导致肺组织NF κB的活化 ,介导炎症介质大量表达 ,参与急性肺损伤发生     

巨噬细胞移动抑制因子在大鼠重症急性胰腺炎肺损伤中作用的实验研究

目的 研究巨噬细胞移动抑制因子(MIF)在大鼠重症急性胰腺炎肺损伤中的作用.方法 雄性SD大鼠,随机分为5组,即正常对照组、SAP3h组、SAP6h组、SAP12h组、SAP24h组,胰胆管逆行注射脱氧胆酸钠,制备大鼠急性胰腺炎模型.检测制模后3h、6h、12h、24h的相关指标:测定血清淀粉酶;胰腺组织和肺组织病理学检查及评分;采用半定量逆转录聚合酶链反应(RT-PCR)检测肺组织MIFmRNA;利用免疫组化方法检测肺组织CD68的表达(反映巨噬细胞数目).结果 实验组大鼠SAP模型在3h时段即有肺损伤表现,并随时间延长而加重.对照组未出现肺损伤表现.实验组肺组织MIFmRNA在制模后3h即明显升高,并维持较高水平,实验组肺组织在3h时段已有巨噬细胞浸润,并一直维持在较高水平,肺组织中MIFmRNA表达与巨噬细胞浸润成正相关(r=0.59,P＜0.05).结论 在SAP肺损伤模型中,表达增多的MIF可能起着重要的介导作用,可以抑制巨噬细胞的移动,并促进其在局部浸润和活化.     

乌司他丁对急性百草枯中毒大鼠肺病理改变的作用研究

目的通过研究乌司他丁（utinastatin,UTI）对急性百草枯（paraquat,PQ）中毒大鼠肺病理学改变的作用研究,探讨乌司他丁对PQ中毒大鼠的肺损伤是否具有保护作用。方法取SD大鼠72只,随机均分为三组,中毒组、乌司他丁组和正常对照组。中毒组及乌司他丁组用百草枯120mg/kg灌胃,一次性染毒;乌司他丁组从染毒后2h开始,腹腔内注射乌司他丁7.5万U/kg,1次/d,至处死前;中毒组及正常对照组用等体积无菌生理盐水腹腔内注射,1次/d,至处死前。分三个不同时间点（6、24、72h）处死大鼠（每组每个时间点8只）,并取肺组织制备标本后分别在光镜及电镜下观察肺组织的病理学改变。结果中毒组大鼠肺的病理学改变为急性弥漫性肺损伤,表现为肺泡腔内出血、渗出,炎性细胞浸润、肺泡隔炎性细胞浸润及纤维组织增生,Ⅰ型和Ⅱ型上皮细胞坏死脱落;乌司他丁组大鼠的病理学改变为局灶性肺泡隔少量炎性细胞浸润,而Ⅰ型上皮细胞完整,Ⅱ型上皮细胞也无明显损伤,未见纤维组织增生。结论从病理学角度观察,乌司他丁对百草枯中毒大鼠的肺损伤具有保护作用。     

钠通道在肺泡液体清除中的作用

肺水增多是急性肺损伤的主要特征.肺泡上皮细胞具有主动转运肺泡液体的作用.最近研究认为,该机制主要和肺泡Ⅱ型上皮细胞顶膜的钠通道主动转运钠离子并随之带动肺泡液体转运有关.探讨钠通道功能调节影响因素及信号转导机制在因急性肺损伤导致的肺水的清除中具有重要的临床意义.     

钙通道与急性肺损伤肺泡Ⅱ型上皮细胞凋亡的研究进展北大核心CSCD

急性肺损伤（ ALI）是重症监护病房最常见的急危重症,由肺内外各种因素引起肺泡上皮细胞（ AEC）和肺毛细血管内皮细胞损伤,造成急性肺水肿,导致顽固性低氧血症,甚者并发严重呼吸衰竭。近年来研究发现,钙离子在细胞凋亡中发挥着重要的作用,本文就钙离子与ALI肺泡Ⅱ型上皮细胞（ AECⅡ）凋亡作以下论述。     

角质细胞生长因子受体转基因对急性肺损伤时肺泡上皮细胞钠离子通道的影响

目的 观察角质细胞生长因子受体(KGFR)腺病毒表达载体在大鼠急性肺损伤前后对肺泡Ⅱ型上皮细胞钠离子通道表达的促进作用,从而验证基因治疗急性肺损伤的效果.方法 将雄性SD大鼠40只分成4组:正常对照组(n=8),致伤对照组(n=10),正常转基因组(n=10),致伤转基因组(n=12).用脂多糖(LPS)造成急性肺损伤模型,以肺组织明显肿大病变为制模成功标准.正常转基因组和致伤转基因组均通过大鼠尾静脉注射KGFR基因的腺病毒表达载体应用液1 mL,72 h后致伤转基因组和致伤对照组分别通过大鼠尾静脉以5 mg/kg注射LPS,正常转基因组和正常对照组则注射等量的生理盐水.过48 h后,断头处死所有实验组大鼠,取肺组织进行实验.通过免疫组化和免疫电镜检测各组大鼠肺泡Ⅱ型上皮细胞钠离子通道的表达情况.数据采用完全随机区组设计的方差分析,行LSD-t检验,以P<0.05为差异具有统计学意义.结果 肺泡Ⅱ型上皮细胞钠离子通道表达水平(免疫组化)由高到低依次为:正常对照组(PU值=47.7±3.33),正常转基因组(PU值=46.9±5.21),致伤转基因组(PU值=29.19±4.11)和致伤对照组(PU值=5.1±2.3);致伤转基因组的钠离子通道表达水平低于正常转基因组(t=9.134,P<0.001)和正常对照组(t=10.601,P<0.001),但明显高于致伤对照组(t=16.466,P<0.001).结论 KGFR腺病毒表达载体转基因效果明显,能够在LPS致伤情况下有效促进钠离子通道的表达,缓解肺泡腔内钠水潴留,达到转基因治疗肺损伤的效果.     

内毒素肺损伤小鼠肺内清道夫受体A表达的变化

目的 观察内毒素肺损伤发病中小鼠肺组织及肺泡巨噬细胞(AM)清道夫受体A(SR-A)表达的变化.方法 腹腔注射内毒素脂多糖(LPS)复制小鼠急性肺损伤模型,实验分为LPS致伤后0.5、1、2、4和8 h组及对照组,用小鼠AM株J774A.1细胞作体外实验,分为LPS作用后0.5、1、2、4和8 h组及无血清培养液对照组.用免疫组化及流式细胞仪观察分析小鼠肺组织及AM、J774A.1细胞的SR-A表达及分布.结果 LPS各组小鼠动脉血氧分压(PaO2)均明显低于对照组,且肺湿/干重(W/D)比值明显高于对照组(P均＜0.01).对照组小鼠肺组织除支气管上皮细胞、淋巴细胞无SR-A表达外,AM、肺血管内皮细胞、血管平滑肌细胞、肺泡上皮细胞、中性粒细胞胞膜及胞质均有SR-A表达.LPS致伤后0.5 h即可观察到肺组织SR-A免疫组化染色弱于对照组,并且随着致伤时间延长,染色逐渐变浅,表明内毒素肺损伤发病中肺内SR-A表达减少.用J774A.1细胞作体外实验也发现类似结果,以4 h和8 h组降低最为显著.流式细胞仪检测AM及J774A.1细胞的SR-A表达与免疫组化染色结果相符,且细胞膜SR-A下降较细胞总SR-A显著.结论 内毒素肺损伤小鼠肺组织及AM的SR-A表达减少,其表达变化可能与内毒素作用有关.     

大黄附子汤对重症急性胰腺炎肺损伤大鼠肺泡上皮屏障功能的影响

目的 探讨大黄附子汤对重症急性胰腺炎肺损伤大鼠肺上皮屏障功能的影响及意义.方法 96只SD大鼠随机分为假手术组、重症急性胰腺炎肺损伤组(SAP-ALI组)及大黄附子汤组三组,每组32只.各组再按3,6,12,24 h时间点分为四个亚组,每个亚组各8只.假手术组开腹后行空肠造瘘,翻动胰腺数次后关腹;SAP-ALI组在假手术组基础上经胰胆管逆行注入4%牛磺胆酸钠(1mL/kg),建立重症急性胰腺炎急性肺损伤模型;大黄附子汤组在SAP-ALI组基础上经空肠造瘘管注入大黄附子汤10 mL.各组按时间点分别于伤后3,6,12,24 h测定肺泡上皮通透性(AEP)、肺泡内液体清除率(ALCR)、总肺水量(TLW)和肺血管外肺水量(EVLW),同时测定血浆内毒素(ET)、血清淀粉酶含量,光镜下观察肺脏病理组织学变化.结果 与假手术组比较,SAP-ALI组大鼠术后3,6,12,24 h ALCR显著降低,TLW,EVLW及AEP呈明显升高趋势(P＜0.05或P＜0.01).与SAP-ALI组比较,大黄附子汤组对大鼠术后3,6,12,24 h ALCR均显著升高,TLW,EVLW及AEP呈明显降低趋势(P＜0.05或P＜0.01),且肺组织的水肿、出血、炎症程度及肺损伤学评分和肺湿/干重比均明显好转.结论 重症急性胰腺炎并急性肺损伤大鼠存在严重的肺泡上皮屏障功能障碍;大黄附子汤通过抑制内毒素过度释放,可降低炎症反应,改善肺泡上皮功能障碍,减轻肺损伤程度.

Abstract：

Objective To investigate effect of Dahuang Fuzi decoction on alveolaur epithelial barrier in rats with lung injury with severe acute pancreatitis. Method Ninty-six health SD rats were randomly divided into three groups: sham operation group, SAP-ALI group, Dahuang Fuzi decoction group, and then according to the time point of sacrifice after operation, each group was subdivided into 3,6,12,24 hour subsets ( each, n = 8). After the belly of a rat in the sham operation group was cut open, the pancreas was flipped several times,and then a stoma was made in the jejunum to form its fistula. In the SAP-ALl group,1 mL/kg sodium taurocholate was reversely injected into the pancreatobile duct to establish the model of SAP, and then the jejimum fistula was performed. The SAP-ALI model in Dahuang Fuzi decoction group was treated by injection of 10ml of Dahuang Fuzi decoctionon into the fistula respectively. Blood was collected from heart to detect serum amvlase and endotoxin (ET) levels before the rat being executed. The lung histopathologic changes, pulmonary injury scores and wet/dry weight(W/D) ratios were observed after the rats were executed. The alveolar liquid clearance rate(ALCR), total lung water content (TLW), extravascular lung water content(EVLW) and alveolar epithelial permeability (AEP) were examined in 3,6, 12,24 h after injury.Results There was continuous increase of AEP,TLW and EVLW,as well as progressive reduction of ALCR compared with sham operation group at 3,6,12,24 h after operation. Compared with SAP-ALI group, there was continuous decrease of AEP,TLW and EVLW, and elevated of ALCR at 3,6,12,24 h after operation.Conclusions Dahuang Fuzi decoction can significantly reduce alveolaur epithelial barrier and degree of lung tissue of SAP-ALI rats by inhibiting the elevation of LPS and inflammation reaction.     

Janus激酶/信号转导和转录激活因子3对重症急性胰腺炎大鼠肺泡Ⅱ型上皮细胞损伤的体外作用研究

目的 探讨Janus激酶/信号转导和转录激活因子3(JAK/STAT3)信号转导通路在重症急性胰腺炎(SAP)肺泡Ⅱ型上皮细胞损伤中的作用.方法 用牛磺胆酸钠建立SAP大鼠模型,取血清备用.将经原代培养的肺泡Ⅱ型上皮细胞随机分组,对照组加入不含SAP大鼠血清的Dulbecco改良Eagle培养基(DMEM)培养液;SAP组加入含SAP大鼠血清的DMEM培养液;SAP+AG490组用JAK激酶抑制剂AG490预处理细胞,加入含SAP大鼠血清的DMEM培养液.用电泳迁移率改变分析法(EMSA)检测STAT3活化状态;逆转录一聚合酶链反应(RT-PCR)检测mRNA表达;蛋白质免疫印迹法(Western blotting)检测蛋白水平;流式细胞技术检测肺泡表面活性物质相关蛋白C(SP-C)表达和肺泡Ⅱ型上皮细胞凋亡.结果 与对照组比较,SAP组STAT3活性增强,STAT3 mRNA和蛋白表达均增强,SP-C蛋白表达下降(2 h SP-C荧光指数0.69±0.02比1.02±0.03,P＜0.01),肺泡Ⅱ型上皮细胞凋亡增加[(11.55±1.10)%比(5.30±0.36)%,P＜0.053;与SAP组比较,SAP+AG490组STAT3活性减弱,STAT3 mRNA和蛋白表达减弱,SP-C蛋白表达下降(2 h SP-C荧光指数0.48±0.10比0.69±0.02,P＜0.01),肺泡Ⅱ型上皮细胞凋亡增加[(13.92±0.82)%比(11.55±1.10)%,P＜0.053.结论 提示JAK/STAT3信号转导通路参与了SAP肺泡Ⅱ型上皮细胞损伤的病理生理过程.     

间质胶原酶在高氧所致急性肺损伤中的表达

目的 研究间质胶原酶(interstitial collagenase)在高氧所致急性肺损伤发病过程中的作用,探讨急性肺损伤的发病机理.方法 将72只C57BL/6小鼠随机分为正常对照组、高氧24 h组、高氧48 h组、高氧72 h组,每组18只;正常对照小鼠呼吸室内空气,高氧小鼠置于密闭的氧气室(氧浓度＞95%).评价肺损伤程度;逆转录-聚合酶链反应(RT-PCR)及免疫组织化学法测定肺组织间质胶原酶的表达及组织分布.统计分析采用单因素方差分析和q检验.结果 高氧能引起急性肺损伤;RT-PCR结果显示肺组织间质胶原酶mRNA表达量在暴露于高氧24 h后达(0.59±0.13)较对照组(0.07±0.01)明显增加(q≥3.15,P＜0.01),72 h达最高(0.68±0.12,q=3.78,P＜0.01);免疫组织化学研究显示间质胶原酶蛋白主要表达于气道上皮细胞、Ⅱ型肺泡上皮细胞和血管平滑肌细胞的胞浆中;间质胶原酶蛋白在气道上皮细胞的表达在高氧环境下24 h达(28.54±9.60)较对照组(13.48±4.32)明显升高(q≥2.62,P＜0.05),于48、72 h表达逐渐降低,分别为(20.32±5.68)和(15.24±4.65).结论 高氧能引起急性肺损伤伴间质胶原酶的表达增高,它们通过降解细胞外基质在高氧所致的急性肺损伤过程中发挥重要作用.     

急性肺损伤幼鼠肺泡Ⅱ型上皮细胞和肺表面活性蛋白A的变化

目的 探索急性肺损伤(ALI)时肺泡Ⅱ型上皮细胞(AEC-Ⅱ)和肺表面活性蛋白A(SP-A)的变化规律.方法 110只SD幼鼠(雄性53只,雌性57只)随机分为对照组、ALI组(每组分6个亚组).ALI组腹腔注射脂多糖(LP3,4 mg/ks),对照组注射生理盐水.于注射后6,12,24,36,48,72 h每组处死8只幼鼠并取材.透射电镜下观察各组AEC-Ⅱ超微结构的变化.采用半定量RT-PCR法测量各组肺组织SP-A mRNA的表达,用Western blot法测量肺组织SP-A含量.采用SPSS 12.0统计软件包进行方差分析和方差齐性检验.结果 注射LPS 24 h后,AEC-Ⅱ表面微绒毛消失.24 h和48 h时AEC-Ⅱ中板层小体(LB)出现暂时性数馈增加.48 h时LB呈特征性"指环状"绕核排列和巨大的空泡样LB.72 h时LB数目显著减少,可见破碎和残余的LB.SP-A从24 h至48 h表达显著增强(P<0.01),于36 h达最高值(6.94±0.80,P<0.01),72 h 下降至最低点(3.87 ±0.50,P<0.01).结论 ALI时AEC-Ⅱ形态变化与肺组织SP-A含量变化是时间依赖性的.AEC-Ⅱ损伤早期伴有肺组织SP-A的代偿性增加.AEC-Ⅱ损伤加重时LB出现特征性变化,可能与SP-A的代偿有关.ALI晚期,AEC-Ⅱ的严重损伤与SP-A的下降密切相关.     

NF-κB和PUMA与重症胰腺炎致急性肺损伤的关系以及PDTC的干预作用

目的 探讨NF-κB(核因子-κB)与PUMA(P53正向凋亡调节因子)表达在大鼠重症急性胰腺炎致急性肺损伤(SAP-ALI)的作用及脯氨酸二硫代氨基甲酸酯(PDTC)的影响.方法 SD大鼠随机(随机数字法)分为假手术组、SAP-ALI组、PDTC组,每组18只.各组再按6,12,24 h时间点分为三个亚组,每个亚组6只.假手术组开腹后翻动胰腺数次;SAP-ALI组采用胰胆管逆行注入5%牛磺胆酸钠(1 mL/kg)诱导SAP-ALI模型;PDTC组在SAP-ALI组的基础于术前1h给予PDTC(15 mg/kg),各组按时间点处死大鼠.观察胰腺和肺脏病理变化.Western-blot法检测肺组织NF-κB p65,PUMA表达,采用RT-PCR法测量各组bax,bcl-2和Caspase-3 mRNA,通过荧光检测试剂检测Caspase-3活性.投射电镜下观察各组肺泡Ⅱ型上皮细胞超微结构的变化,TUNEL检测肺泡上皮细胞的凋亡指数.结果 成功建立了大鼠SAP-ALI模型,Western-blotting结果显示,SAP-ALI组肺组织NF-κB p65和PUMA蛋白表达在6h后增加,24 h增加最明显,NF-κB p65与PUMA显著正相关;SAP-ALI组肺组织bax mRNA和Caspase-3mRNA在6 h后增加,24 h增加最明显,bcl-2 mRNA在6 h后下降,24 h下降最明显,Caspase-3活性在24h增加最明显.PDTC组肺组织NF-κB p65和PUMA蛋白表达明显降低;bax mRNA和caspase-3 mRNA表达及Caspase-3活性明显降低,bcl-2 mRNA表达明显增加.假手术组肺组织各组蛋白的表达与PDTC组比无显著改变.PDTC组肺损伤病理组织学评分在术后各时间点较SAP-ALI组显著降低,PDTC组细胞凋亡指数较SAP-ALI组明显降低.SAP-ALI组24h市泡Ⅱ型上皮细胞微绒毛消失.结论 NF-κB活化激活PUMA与肺泡上皮细胞凋亡有关,PDTC通过抑制NF-κB活化,下调NF-κB活化后引起的PUMA表达,抑制肺泡Ⅱ型上皮细胞凋亡,减轻SAP-ALI.     

肺泡中肺表面活性蛋白A反映冲击伤复合缺氧的肺损伤程度

目的 研究冲击伤及冲击伤复合缺氧时支气管肺泡灌洗液(BALF)中肺表面活性蛋白A(SP-A)含量变化与肺损伤的关系及意义.方法 Wistar大鼠131只(野战外科研究所实验动物中心提供),随机分为4组:冲击伤(BI)组47只,冲击伤复合缺氧Ⅰ组(BAⅠ)36只,冲击伤复合缺氧Ⅱ组(BAⅡ)40只,正常对照(Nc)组8只.用BST-Ⅰ型生物激波管致冲击伤,BAⅠ、BAⅡ组伤后立即置入低氧舱内,分别通入含12.5%和10%的O2的混合气体,并分别保持舱内氧的体积分数稳定在12.5%和10%.NC组大鼠不做任何处理.动物分别于伤后1、3、6 h活杀,行大体解剖、光镜和电镜病理观察、肺含水量测定,Western blot法测定BALF中SP-A含量.计量资料数据以均数±标准差(x±s)表示,采用组间比较q检验和相关分析;计数资料采用x2检验.结果 大鼠冲击伤后呼吸加快,BA Ⅰ组和BA Ⅱ组有烦躁不安、呼吸急促,BAⅡ组可见明显口鼻发绀;致伤各组肺含水率明显高于正常大鼠(P<0.05).肺大体解剖主要表现为肺出血、肺水肿,光镜下见肺泡壁增厚、肺泡腔出血、间质水肿;电镜下可见肺泡壁断裂,Ⅱ型上皮细胞板层体减少;这些改变均以 BAⅡ组最重、BA Ⅰ次之、BI再次,6 h的死亡率分别为37.5%、11.1%和2.1%(P<0.01).伤后BALF中SP-A浓度显著降低(P<0.01),并与肺含水率呈显著负相关(r=-0.796,P<0.01).结论 冲击伤复合缺氧显著加重肺部的损伤,缺氧越重、时间越长,则肺损伤越重、死亡率越高;BALF中SP-A含量的显著减少与肺水肿程度有关,BALF中SP-A含量可作为判断冲击伤及其复合伤肺损伤严重程度的观察指标.     

Neutrophils and their Fc gamma receptors are essential in a mouse model of transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is the most common cause of transfusion-related mortality. To explore the pathogenesis of TRALI, we developed an in vivo mouse model based on the passive transfusion of an MHC class I (MHC I) mAb (H2Kd) to mice with the cognate antigen. Transfusion of the MHC I mAb to BALB/c mice produced acute lung injury with increased excess lung water, increased lung vascular and lung epithelial permeability to protein, and decreased alveolar fluid clearance. There was 50% mortality at a 2-hour time point after Ab administration. Pulmonary histology and immunohistochemistry revealed prominent neutrophil sequestration in the lung microvasculature that occurred concomitantly with acute peripheral blood neutropenia, all within 2 hours of administration of the mAb. Depletion of neutrophils by injection of anti-granulocyte mAb Gr-1 protected mice from lung injury following MHC I mAb challenge. FcRgamma-/- mice were resistant to MHC I mAb-induced lung injury, while adoptive transfer of wild-type neutrophils into the FcRgamma-/- animals restored lung injury following MHC I mAb challenge. In conclusion, in a clinically relevant in vivo mouse model of TRALI using an MHC I mAb, the mechanism of lung injury was dependent on neutrophils and their Fc gamma receptors.     

Ⅱ型减压病肺组织超微结构改变

目的观察兔实验型减压病肺组织超微结构改变,探讨减压病对肺组织损伤的机制。方法用雄性新西兰家兔制备减压病模型。动物出舱后30～60分钟内处死,取肺尖部组织,制备超薄切片,电镜下观察超微结构。并设安全减压组和正常组作为对照。结果减压病组：电镜下见肺泡腔部分闭塞,部分实性变,内见红细胞渗出,I型肺泡上皮细胞突起回缩,胞体肿胀,线粒体肿胀。Ⅱ型肺泡上皮细胞肿胀明显,细胞质内线粒体扩张,板层小体减少;另见部分Ⅱ型上皮细胞固缩,核染色质凝集,固缩的细胞内线粒体空泡变性,板层小体消失。毛细血管扩张充血,毛细血管内皮细胞肿胀,基底膜不完整。安全减压组：部分肺泡腔扩大,毛细血管充血,I性肺泡上皮和毛细血管内皮细胞轻度肿胀;Ⅱ型肺泡上皮线粒体轻度肿胀,溶酶体数目增多,板层小体细胞膜下聚集。结论减压病可导致呼吸上皮损伤、血气屏障破坏,影响肺泡气体交换;其机制可能与减压过程中,在血管和组织内产生的游离气泡有关。     

Expression of phosphatidylinositol-3 kinase and effects of inhibitor Wortmannin on expression of tumor necrosis factor-α in severe acute pancreatitis associated with acute lung injury

BACKGROUND: Acute lung injury(ALI) is a common and serious complication of severe acute pancreatitis(SAP). The study aimed to investigate the protective effect and mechanism of phosphatidylinositol-3 kinase(PI3K) inhibitor Wortmannin in SAP associated with ALI.METHODS: Ninety rats were randomly divided into three groups: sham operation(SO) group(n=30), SAP group(n=30), and SAP+Wortmannin(SAP+W) group(n=30). SAP model was induced by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct of rats. The rate of lung water content, myeloperoxidase(MPO), matrix metalloproteinase 9(MMP-9), protein kinase B(PKB), abdphosphorylation of protein kinase B(P-PKB) activity in the lung tissue were evaluated.RESULTS: In the SAP group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours(P0.05); the rate of lung water content, MPO and TNF-α activity were also gradually increased, and the degree of lung lesion gradually increased(P0.05). In the SAP+Wortmannin group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours; it was higher than that in the SO group(P0.05), but significantly lower than that in the SAP group(P0.05). The rest indicators in the SAP+Wortmannin group were also significantly decreased as compared with the SAP group(P0.05).CONCLUSIONS: The expression of phosphatidylinositol-3 kinase/protein kinase B was elevated in severe pancreatitis rats with lung injury. This suggested that PI3 K signal transduction pathway is involved in the control and release of proinfl ammatory cytokines TNF-α, which may play an important role in the pathogenesis of severe acute pancreatitis associated with lung injury. This finding indicated that Wortmannin can block the PI3 K signal transduction pathway, and inhibit the release of infl ammatory factor TNF-α.     

Lung fluid transport in aquaporin-5 knockout mice

The mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia, AQP4 in airway epithelia, and AQP5 at the apical plasma membrane in type I cells of alveolar epithelia. We previously studied the role of AQP1 and AQP4 in lung fluid transport using knockout mice. Here, we examined the role of AQP5 using AQP5 knockout mice, which were recently shown to manifest defective saliva secretion. AQP5 deletion did not affect lung morphology at the light microscopic level, nor did it affect the distribution or expression of aquaporins 1, 3, or 4. Airspace-capillary osmotic water permeability (P_f) was measured in isolated perfused lungs by pleural surface fluorescence and gravimetric methods. P_f was reduced 10-fold by AQP5 deletion and was further reduced by 2- to 3-fold in AQP1/AQP5 double-knockout mice. Hydrostatic lung edema in response to acute increases in pulmonary artery pressure was not affected by AQP5 deletion. Active alveolar fluid absorption was measured in an in situ lung model from the increase in concentration of a volume marker in an isosmolar alveolar instillate. Interestingly, fluid absorption did not differ in litter-matched AQP5 knockout mice, nor was there an effect of AQP5 deletion when fluid absorption was maximally stimulated by pretreatment of mice with keratinocyte growth factor. These results indicate that AQP5 is responsible for the majority of water transport across the apical membrane of type I alveolar epithelial cells. The unimpaired alveolar fluid clearance in AQP5-null mice indicates that high alveolar water permeability is not required for active, near-isosmolar fluid transport.