【作者】 罗怀安；

【导师】 张海鹏；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 前言在休克、胰腺手术、胰腺移植中,胰腺的缺血再灌注(I／R)损伤仍然是一个重要的临床问题。损伤的主要机制是产生大量氧自由基和缺血性炎症。许多研究表明,胰腺的I／R能增加血中白细胞数,氧自由基的生产,及细胞因子的释放,从而引起急性胰腺炎及全身炎症反应综合症。在全身炎症反应综合征中肺脏是首位受累的靶器官。因为,肺脏是唯一接受全部心脏排出量的器官,受循环中炎性细胞及介质的损伤最大,隔离在肺部的活化炎性细胞和炎性胰腺释放的蛋白酶都会诱发急性肺损伤。急性胰腺炎是胰酶在胰腺内被激活后引起胰腺组织自身消化的一种炎症反应性疾病,主要表现为血清淀粉酶和脂肪酶升高。急性胰腺炎相关性肺损伤(acute pancreatitis-associated lung injury, APALI)发病机制复杂,可通过某种机制导致胰腺酶的不适当激活,这种机制包括酶解作用衍生的催化剂激活炎性细胞,白细胞的释放,及氧化和亚硝化应激的发生,从而改变气道反应性。目前研究认为核因子κB(nuclear factor-KB)在其中扮演重要角色,其活化被认为是急性胰腺炎重要的早期事件。(NF-κB)是一种转录调节因子,在细胞因子介导的感染、炎症反应、氧化应激、细胞增生、细胞凋亡等过程中起重要作用。正常生理情况下,NF-κB以无活性的形式存在于多种细胞的胞质中,激活后促进多种细胞因子的基因转录,在炎症反应复杂的细胞因子网络中,NF-κB的活化可能是一个中心环节,研究表明NF-κB通过促进TNF-α、IL-6、IL-8、细胞间黏附分子(Intercellular adhesion molecular, ICAM)等基因的转录而参与肺损伤的发生,其中ICAM-1在胰腺炎引起的肺损伤中最受关注。ICAM-1属于免疫球蛋白超家族成员,其在人体内的分布十分广泛,炎症介质能明显上调血管内皮细胞和其它非造血细胞ICAM-1的表达。肺血管内皮上表达的ICAM-1结合活化的白细胞表面的整合素CDllb／8β是白细胞的黏附、游走、聚集过程中的关键环节,其过度表达可以促进局部炎性反应发生。巨噬细胞的作用越来越引起人们的重视。近年研究表明巨噬细胞活化可能是急性胰腺炎时发生肺损伤的的重要途径之一。活化的巨噬细胞可以释放许多生物活性物质,如细胞因子、花生四烯酸等,均为前炎性反应介质,可以介导PMN等释放多种炎症反应介质。巨噬细胞移动抑制因子(macro-phage migration inhibitory facter, MIF)具有抑制巨噬细胞游走,促进巨噬细胞的黏附和在炎症局部浸润的作用,并可刺激炎症细胞分泌TNF、IL-1等促炎性细胞因子。巨噬细胞炎症蛋-2(macrophage inflammatory protein 2, MIP-2)是大鼠ELR+(含谷-亮-精氨酸功能基序)CXC类趋化性细胞因子,在功能上和人类IL-8同源,是中性粒细胞的主要趋化细胞因子。本研究通过大鼠胰腺缺血再灌注模型,探讨大鼠胰腺缺血再灌注时,合并肺损伤、诱导气道高反应性中的作用；并探讨NF-κB与ICAM-1mRNA表达及MIF与MIP-2在胰腺缺血再灌注并发肺损伤中的作用。材料与方法一、动物模型和样品制备1、I／R动物模型通过阻断胃十二指肠动脉和脾动脉2小时,再灌注6小时诱导胰腺缺血。假手术组以相同的手术方法切开显露胃十二指肠动脉和脾动脉,但不夹闭血管。2、试验取材取右股静脉血作为血样。实验结束时向肺内注入5ml生理盐水,获取肺灌洗液。Sham组没有阻断动脉,其值作为未阻断的基础对照值。切取肺组织,-80℃冷冻保存。二、观察指标及测定方法1、胰腺缺血再灌注诱导的气道高反应性研究(1)收集血液样本离心后,使用Kodak Ektachem DT60分析器(罗切斯特,纽约)测量血浆中分离的淀粉酶含量,以IU／L表示。(2)高效液相色谱法测量血液中源自一氧化氮(NO)的亚硝酸盐和硝酸盐阴离子(3)通过分光荧光计测量血液中甲基胍。(4)白细胞计数测量肺灌洗液标本中的WBC。(5)通过酶联免疫测定血液中肿瘤坏死因子(TNF-α)的表达量。按试剂盒说明书进行操作。显色后用酶标仪(波长450nm)比色读数,根据标准曲线求出TNF-α数值。(6)全身体积描记法(Buxco co)测定气道对乙酰甲胆碱的反应变化。双室体描仪由头室和体室组成,各置一流量传感器,分别用于测量鼻部呼吸引起的气流变化和胸廓运动引起的气流变化。流量传感器感受到的流量变化转变成电信号,经放大器放大,转换成数字信号后,通过软件(BioSystem XA software with NAM analyzer)分析,计算出大鼠气道基线增强暂停系数(the baseline enhanced pause, Penh)。(7)实时监测PCR采用mRNA分离试剂盒分离肺组织中的mRNA;使用ABI公司7000型检测棱镜(应用生物系统公司)实时监测PCR扩增反应。通过实时聚合酶链反应测定肺组织中的iNOS的mRNA表达和肿瘤坏死因子(TNF-α)的表达。2、NF-κB与ICAM-1在I／R并发肺损伤的作用研究(1)组织病理学评分：取各组大鼠胰头部组织和右肺下叶组织经4%多聚甲醛固定、石蜡包埋、HE染色,光镜观察组织病理学变化并进行评分。(2)收集血液样本离心后,使用Kodak Ektachem DT60分析器(罗切斯特,纽约)测量血浆中分离的淀粉酶含量,以IU／L表示。(3)肺组织MPO检测按照试剂盒说明书操作。将肺组织机械匀浆后水浴、比色、参照如下公式计算：MPO(U／g)湿片=(测定管OD值-对照组OD值)／11.3×取样量(g)(4) Western Blot法检测肺组织ICAM-1蛋白表达,凝胶成像系统对结果照相及测定条带的面积和灰度值,以目的条带的面积×灰度值/Actin条带的面积×灰度值的比值代表蛋白的表达水平。(5)NF-κB相对活性检测：结果用Leica Q500Mc图像分析系统进行密度分析,以灰度值表示NF-κB相对活性变化。3、I／R并发肺损伤中MIF与MIP-2的表达及意义研究(1)组织病理学评分：取各组大鼠胰头部组织和右肺下叶组织经4%多聚甲醛固定、石蜡包埋、HE染色,光镜观察组织病理学变化并进行评分。(2)收集血液样本离心后,使用Kodak Ektachem DT60分析器(罗切斯特,纽约)测量血浆中分离的淀粉酶含量,以IU／L表示。(3)肺组织MPO检测按照试剂盒说明书操作。将肺组织机械匀浆后水浴、比色、参照如下公式计算：MPO(U／g)湿片=(测定管OD值-对照组OD值)／11.3×取样量(g)(4)RT-PCR法检测肺组织MIF mRNA的表达,采用Trizol一步法提取肺组织总RNA,紫外分光光度仪测定RNA浓度。用TC 21000数据图像分析系统分析各条带灰度值,得MIF/GADPH的灰度比值,即为MIF mRNA的相对表达值。(5)肺组织MIP-2含量测定：肺组织用10倍体积的预冷匀浆介质制成匀浆,一份用ELISA法检测MIP-2浓度,采用ELISA全自动检测仪按rMIP-2/GRO-βELISA试剂盒供应商提供的说明书设定反应步骤；一份用全自动生化分析仪测定蛋白含量。4、统计学分析采用SPSS 13.0软件进行统计学分析。对数据进行正态性检验后,用均数(Mean)和标准差(SD)描述正态分布数据的集中趋势和离散水平。组间各检测指标比较采用t检验,试验前后比较采用配对t检验,p值<0.05认为有统计学意义。结果1、胰腺缺血再灌注诱导的气道高反应性研究本实验研究发现I／R组小鼠血中的一氧化氮,羟自由基,淀粉酶,肿瘤坏死因子,白细胞浓度的显著升高。在缺血再灌注(I／R)后肺组织中iNOS和肿瘤坏死因子的mRNA的表达明显增加,肺功能的数据显示,胰腺的缺血／再灌注(I／R)诱导气道对乙酰甲胆碱的反应大量增加；与假手术组相比,I／R组中的PenH显著增加,而且灌洗液白细胞明显增加。2、NF-κB与ICAM-1在I／R并发肺损伤的作用研究I／R组大鼠胰腺和肺组织病理学评分分别为5.94±0.72和6.42±0.65；显著的高于Sham组大鼠病理评分(分别为：0.20±0.14和0.27±0.31)(p<0.05)；Sham组大鼠血清中淀粉酶的水平为1198.4±121.7；I／R组大鼠血清中淀粉酶的水平为3719.6±523.8；两组间差异达到统计学意义(p<0.05)。与Sham组大鼠比较,胰腺缺血再灌注可显著的增高肺组织中MPO水平的表达(0.74±0.06)(p<0.05)；Sham组大鼠肺组织中ICAM-1蛋白和ICAM-1 mRNA微弱表达,I／R组大鼠肺组织中ICAM-1蛋白和ICAM-1 mRNA表达增高,其表达水平分别为0.47±0.03和1.12±0.07；胰腺缺血再灌注后可使大鼠肺组织中NF-κB活性水平显著的增高。3、I／R并发肺损伤中MIF与MIP-2的表达及意义研究Sham组胰腺组织和肺组织病理评分分别为2.14±0.06和0.37±0.14；I／R组胰腺组织和肺组织病理评分分别为8.52±1.17和4.71±0.30；两组间病理评分差异达到统计学意义(p<0.05)。胰腺缺血再灌注可显著的增高血清中淀粉酶和肺组织中MPO水平的表达。sham组大鼠血清中MIF和肺组织中MIFmRNA表达微弱,,而I／R组大鼠血清中MIF和肺组织中MIFmRNA表达增高。Sham组大鼠肺组织中MIP-2活性水平为23.9±5.8；胰腺缺血再灌注后可使大鼠肺组织中MIP-2活性水平显著的增高(91.5±12.1)。结论1、胰腺I／R诱导全身炎症反应及肺内白细胞(WBC)的增加。再灌注组中气道的高反应性可能是因为气道炎症,后者增加肺内WBC的聚集及肺组织中iNOS的表达、肿瘤坏死因子、炎症介质的表达。2、胰腺缺血再灌注损伤可增高肺组织ICAM-1蛋白和ICAM-1基因水平表达。胰腺缺血2小时再灌注6小时后肺组织NF-κB水平增高。3、胰腺缺血再灌注损伤可增高血清和肺组织MIF和MIFmRNA基因水平表达。肺组织MIP-2含量增高,作为早期的促炎性细胞因子,MIP-2介导了肺损伤。更多还原

【Abstract】 IntroductionIschemia-Reperfusion (I/R) injury to the pancreas remains an important clinical problem during shock, pancreatic surgery, and pancreas transplantation. Oxygen free radicals are involved in I/R-related pancreatic injury. Many studies have demonstrated that I/R of the pancreas induce systemic inflammatory responses by increasing the blood white blood cell count, oxygen radical production, and cytokine release. Acute pancreatitis can lead to inappropriate activation of pancreatic enzymes through a mechanism involving proteolytically derived activators by which inflammatory cells are activated, interleukin released, oxidative and nitrosative stress occur, changing airway reactivity. In this study, we characterized whether I/R of the pancreas induced inflammation reactivity changes in airways upon challenge with a cholinergic agonist.Some reports have indicated that intercellular adhesion molecule-1 (ICAM-1) plays an important role in the development and progression of acute pancreatitis complicated by acute lung injury, and the severity of the lung injury correlates well with the expression levels of ICAM-1 protein. ICAM-1, a single-chain transmembrane glycoprotein with a molecular weight of 80-110 KDa, consists of five Ig-like domains, a hydrophobic transmembrane domain and a short cytoplasmic C-terminal domain. Its ligand includes lymphocyte function-associated antigen-1 (LFA-1) and macrophage antigen-1 (Mac-1). Under physiological conditions, ICAM-1 is expressed at a low level in endothelial cells and epithelial cells or constitutively on the surface of alveolar cells, providing the underlying molecular basis for cell recognition, activation, proliferation, differentiation and motility, and thereby helping to stabilize the internal environment of the body. Moreover, ICAM-1 also plays a key role during pathological conditions, such as inflammatory reaction etc. For these reasons, a comprehensive and objective understanding of ICAM-1 is needed. It is obvious that NF-kappa B plays a critical role in the expression of ICAM-1. Therefore, research on the use of NF-kappa B inhibitor to alleviate inflammation response has become a hotspot. Calpain I inhibitor and pyrrolidine dithiocarbamate (PDTC) are antioxidants which are potent inhibitors of NF-kappa B. Calpain I inhibitor and pyrrolidine dithiocarbamate (PDTC) can lessen lung injury in rats with acute pancreatitis, decrease the activation of NF-kappa B as well as the expression of ICAM-1 protein, and can retain the soakage of inflammatory cells and mitigate the microvascular impairment of the lungs, which reduces the incidence rate of pneumonedema. After Hietaranta et al. first reported that MG132, a prosome inhibitor, could depress the activation of NF-kappa B in acute pancreatitis, some researchers demonstrated that MG132 also had the effect of protecting lung tissue in rats with acute pancreatitis which may be associated with the function of inhibiting NF-kappa B activation. The use of the NF-kappa B inhibitor may be considered as another effective path in the treatment of acute pancreatitis complicated by acute lung injury, and associated clinical research is required.MIF was originally identified as a cytokine derived from activated T cells. However, MIF is now considered to exert various biologic functions in macrophage activation. Moreover, MIF is thought to play a central role in exacerbation of inflammation and sepsis. Importantly, a recent report has suggested that gene expression of TLR-4 in macrophages can be upregulated by MIF. Thus, hyporesponsiveness of MIF-deficient macrophages to LPS has been demonstrated by a marked reduction in the activity of NF-κB and the production of TNF-a, which is strictly associated with downregulation of TLR-4. During AP, MIP-2 is involved in neutrophil activation and sequestration in the pancreas and lungs. MIP-2 is a potent rodent chemokine, homologous to GRO-b, which binds to the C-X-C chemokine receptor-2. We found that cerulein induced AP was associated with a significant increase in serum MIP-2 concentrations, and that leptin treatment substantially decreased the MIP-2 concentration. The effect of leptin administration on serum MIP-2 concentration is not clearly established. To the best of our knowledge, this is the first study demonstrating the effect of leptin on serum MIP-2 concentration. A decrease in the concentration of MIP-2 may therefore play an important role in reducing neutrophil adhesion and sequestration. Moreover, Ob-R is present in the pancreas and lungs.Material and Methods1. Animal samples (1) Experimental DesignAnimals were randomly divided into two groups:(1) The I/R group (n=8) underwent 2 hours of gastroduodenal and splenic artery occlusions followed by 6 hours of reperfusion. The rats were not given treatment except saline prior to clamping the arteries. (2) The sham group hosts (n=7) were prepared in the same manner as in the I/R group, but the vessels were not clamped.(2) Preparation of AnimalsMale Sprague-Dawley rats (300-350 g) were anesthetized with pentobarbitals the right femoral vein was cannulated for blood sampling. The gastroduodenal artery and the splenic artery were exposed and ischemia induced by clamping for 2 hours followed by 6-hour reperfusion.2. Experimental Methods(1) Quantification of Pancreatitis by Measuring Plasma Activities of AmylaseBlood samples were collected for WBC measurement. After centrifugation, plasma was isolated for amylase measurement using a Kodak Ektachem DT60 analyzer (Rochester, NY) and expressed in IU/L.(2) Measurement of Nitric Oxide by High-Performance Liquid ChromatographyHigh-performance liquid chromatography was used to measure blood levels of nitrite and nitrate anions derived from nitric oxide (NO).(3) Methylguanidine Measured by SpectrofluorometerWe measured the levels of methylguanidine in blood as a reflection of I/R-induced hydroxyl radical production.(4) WBC Counts in Lung LavagesLung lavages were performed with 5 mL saline at the end of the experiment. WBC in lavage samples were measured using a cell counter.(5) Quantitation of Tumor Necrosis Factor-a by Enzyme-Linked lmmunosorbent AssayThe tumor necrosis factor-a (TNFa) concentrations in blood samples were measured separately with an enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions (Endogen, Woburn, MA).(6) Measurement of Bronchial Responsiveness to MethacholineAirway responses to methacholine challenge were measured by unrestrained. whole-body plethysmography (Buxco Co). Rats placed inside Plexiglas chambers underwent measurements of their respiratory rate and breathing volume using pressuresensitive transducers that had been calibrated for the experimental conditions. After an acclimation period of about 10 minutes, the baseline enhanced pause (Penh), a measure of airway resistance, was determined by exposing the animals to a saline aerosol and calculating Penh according to an algorithm developed by Buxco. Methacholine at specifically metered dose rates was then fed into the chambers and Penh measured again.(7) RNA Isolation and Real-Time Polymerase Chain ReactionIsolation of mRNA from lung tissues was performed using an mRNA Isolation Kit (QIAGEN RNeasy kits, QIAGEN Inc, Valencia, CA). The mRNA isolated from each lung tissue sample was reversely transcribed to cDNA following the manufacturer’s recommendation. Polymerase chain reaction (PCR) primers and TaqMan-MGB probes were designed using Primer Express V.2.0 software (Applied Biosystems Inc, Foster City, CA) based on sequences from GenBank. TaqMan-MGB probes were labeled with 6-carboxy-fluorescein as the reporter dye. PCR reactions were monitored in real time using an ABI PRISM 7000 Sequence Detector (Applied Biosystems Inc).(8) ICAM-1 expression in lung tissue by Western blotWestern blot analysis was performed as described previously. Briefly, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE; 12% separating, 4% stacking) and transferred to NC membranes (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). After the membranes were blocked in 5% nonfat dry milk in PBS buffer containing 0.1%, the protein signal was amplified and visualized via chemiluminescence using the ECL Western blotting detection system and Hyperfilm ECL autoradiograhpy film (Amersham Pharmacia Biotech, Inc.). Images were quantified using the Labworks v3.0.2 image scanning and analysis software.(8) Total MIF mRNA isolation and real time RT-PCRTotal RNA was isolated using the acid guanidinium thiocyanate-phenol-chloroform method. The quality and quantity of the isolated RNA was determined before using the RNA. One microgram of total RNA was reverse transcribed using Advantage RT-for-PCR kit. Real-time RT-PCR was done using Smart Cycler (Cepheid, Sunnyvale, CA) in which 2μl cDNA,10μl Sybergreen Master mix, and 0.5μlof 20 μM gene-specific primers were used. The specificity and size of the PCR products were tested by adding a melt curve at the end of the amplifications and running it on a 2% agarose gel. All values were normalized to 18S expression.(9) Level of MP-2 in Lung tissueConcentrations of MIP-2 in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits.3. Data AnalysisData were expressed as mean values±standard errors of the means. Comparisons within each group for a given parameter used paired Student t tests. Values of P<0.05 were considered statistically significant.Results1.Ischemia-Reperfusion of the Pancreas Induced Hyperresponsiveness of the Airways in RatsThis protocol resulted in significant elevations of the blood concentrations of nitric oxide, hydroxyl radical, amylase, TNFa, and white cells among the I/R group. The mRNA expressions of iNOS and of TNFαin the lung tissues were significantly increased after I/R. Pulmonary function data showed that I/R of the pancreas induced significant increases in the responses to methacholine challenge:Penh was significantly increased in the I/R group compared with the sham group. Lavage white cells were significantly increased in the I/R group.2. Efect of NF-κB and Intercellular Adhesion Molecule-1 on Ischemia-Reperfusion of the Pancreas Associated Lung Injury in RatsThe histologic scores of pancreas and lung in I/R group were 5.94±0.72 and 6.42±0.65 respectively, which was significantly higher than that in sham group (0.20±0.14 and 0.27±0.31, respectively)(p<0.05). The levels of amylase and myeloperoxidase activity were increase significantly in I/R group (3719.6±523.8, 0.74±0.06, respectively) than in sham group (1198.4±121.7). The overexpression of ICAM-1 protein and mRNA level was related with lung injury in I/R rats (0.47±0.03 and 1.12±0.07), comparing with the control group. The level of NF-κB activity also significantly increased. 3. Role of Macrophage Inflammatory protein 2 and Macrophage Migration Inhibitory Factor in Ischemia-Reperfusion of the Pancreas-associated Lung InjuryThe histologic scores of pancreas and lung in I/R group were 8.52±1.17 and 4.71±0.30 respectively, which was significantly higher than that in sham group (2.14±0.06 and 0.37±0.14, respectively) (p< 0.05).The levels of amylase and myeloperoxidase activity were increase significantly in I/R group. The overexpression of MIF protein and mRNA level was related with lung injury in I/R rats comparing with the control group. The level of MIP-2 in I/R group (91.5±12.1) was significantly hisher than that in sham group (23.9±5.8) (p< 0.05).ConclusionI/R of the pancreas induced systemic inflammatory responses and increased white cell sequestration in the lung. Hyperresponsive responses in the airways of the reperfusion group may be due to airways inflammation, which increased white cell sequesteration in the lung and the expressions iNOS and TNF-a inflammatory mediators in lung tissues.更多还原