胰腺缺血再灌注诱导大鼠肺损伤的机理研究

发布时间：2018-04-08 11:14

本文选题：胰腺　切入点：缺血再灌注　出处：《中国医科大学》2010年博士论文

【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)(p0.05)；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(p0.05)。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)(p0.05)；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(p0.05)。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。
[Abstract]:Preface
In shock, pancreatic surgery, transplantation of pancreas, pancreatic ischemia reperfusion injury (I / R) is still a major clinical problem. The main mechanism of injury is to generate a large number of oxygen free radicals and ischemic inflammation. Many studies show that pancreatic I / R can increase the number of white blood cells in the blood, oxygen free radical the production and release of cytokines, which cause acute pancreatitis and systemic inflammatory response syndrome.
In systemic inflammatory response syndrome in the lung is the target organ involvement. First because the lung is only acceptable for all cardiac output organs, by circulating inflammatory cells in the medium and the maximum damage isolation will induce acute lung injury in the activation of inflammatory cells and pancreatic proteases released from the lungs. Acute pancreatitis is a kind of inflammatory disease in the pancreas trypsin activated pancreatic tissue by their own digestion, mainly for serum amylase and serum lipase. Acute pancreatitis associated lung injury (acute pancreatitis-associated lung injury, APALI) complex pathogenesis, inappropriate activation of pancreatic enzymes can be caused by some mechanism, this mechanism comprises a catalyst derived enzymatic activation of inflammatory cells, white blood cells release and oxidative and nitrosative stress, thus changing the airway reactivity.
The present study of nuclear factor kappa B (nuclear factor-KB) which plays an important role in the activation, is considered to be an important early event in acute pancreatitis. (NF- B) is a transcription factor, cytokine mediated infection, inflammation, oxidative stress, cell proliferation, plays an important role in the process of cell apoptosis. Under normal physiological conditions, cytoplasmic NF- kappa B exists in the form of activity in a variety of cells, cytokines promote gene transcription after activation, in the network of inflammatory cytokines in complex NF- kappa B activation may be a key link, studies show that NF- kappa B through promotion of TNF- alpha, IL-6, IL-8, intercellular adhesion molecule (Intercellular adhesion, molecular, ICAM) gene transcription in lung injury, lung injury in the ICAM-1 pancreatitis caused by the most popular.ICAM-1 belongs to the immunoglobulin super family Family members, it is widely distributed in the human body, inflammatory mediators could significantly up regulate the expression of vascular endothelial cells and other non hematopoietic cells. The expression of ICAM-1 in pulmonary vascular endothelial ICAM-1 on the surface of activated white blood cells with CDllb / beta 8 integrin adhesion, leukocyte migration, aggregation of key links in the process of the overexpression can promote the occurrence of local inflammatory reaction.
More and more people pay attention to macrophages. Recent studies suggest that macrophage activation may be an important pathway of lung injury in acute pancreatitis. Activated macrophages can release many bioactive substances, such as cytokines, four arachidonic acid, are proinflammatory mediators, can mediate PMN release various inflammatory reaction medium. Macrophage migration inhibitory factor (macro-phage migration inhibitory facter, MIF) can inhibit macrophage migration, promote macrophage adhesion and infiltration in inflammation, and inflammatory cells can stimulate the secretion of TNF, IL-1 and proinflammatory cytokines. Inflammatory macrophages (macrophage inflammatory egg -2 protein 2, MIP-2 (ELR+) rat Hangu bright arginine motifs) CXC chemotactic cytokines, in function and human IL-8 is homologous, neutrophil The main chemotactic cytokines. The ischemia reperfusion model of ischemia reperfusion, with lung injury, induced airway hyperresponsiveness in action; and to explore the role of NF- K B and the expression of ICAM-1mRNA and MIF and MIP-2 in pancreatic ischemia reperfusion pulmonary injury.
Materials and methods
An animal model, and sample preparation
1, I / R animal model
By blocking the gastroduodenal artery and the splenic artery for 2 hours and 6 hours of reperfusion induced pancreatic ischemia. Sham operation group with the same surgical incision and exposure of the gastroduodenal artery and the splenic artery, but without occlusion of blood vessels.
2, test materials
Take the right femoral vein as the blood samples. 5ml saline was injected into the lungs to the end of the experiment, obtain the lung lavage fluid.Sham group without artery occlusion, its value as a basis for not blocking the control value. Cut the lung tissue, freezing storage at -80.
Two, observation index and determination method
1, study on airway hyperresponsiveness induced by ischemia reperfusion of pancreas
(1) blood samples were collected after centrifugation, using Kodak Ektachem DT60 analyzer (Rochester, New York) amylase content in plasma separation and measurement, using IU / L.
(2) HPLC measurement of blood from nitric oxide (NO) nitrite and nitrate anions
(3) by spectrofluorometer. Measurement of blood in methyl guanidine
(4) white blood cell count measurement of lung lavage fluid specimens in WBC.
(5) determination of blood tumor necrosis factor by ELISA (TNF- alpha) expression. According to the operation instructions Kit. After coloration with a microplate reader (wavelength 450nm) colorimetric readings, TNF- alpha numerical calculated according to the standard curve.
(6) whole-body plethysmography (Buxco CO) determination of airway reactivity to methacholine changes. Using double chamber plethysmography chamber and head body chambers, each provided with a flow sensor for measuring changes in nasal airflow were caused by respiratory airflow changes and thoracic movement caused by changes in the flow sensor flow change. Is feeling into electrical signals, amplified by the amplifier, after converted into digital signal by software (BioSystem XA software with NAM analyzer) analysis, calculate the baseline airway increased in rats (the baseline enhanced pause suspended coefficient, Penh).
(7) real-time monitoring of PCR using mRNA Isolation Kit from the lung tissue of mRNA; using ABI type 7000 prism detection (application Bio System Inc) amplification for real-time monitoring of PCR. The expression of mRNA and tumor necrosis factor iNOS in lung tissue were measured by real-time polymerase chain reaction (TNF- alpha) expression.
2, NF- kappa B and role of ICAM-1 in I / R complicated with lung injury
(1) histopathological scores of rats in each group: take the head of the pancreas tissue and the lower lobe of the right lung tissue was fixed by 4% paraformaldehyde, embedded in paraffin, HE staining to observe the pathological changes and evaluated by light microscope.
(2) blood samples were collected after centrifugation, using Kodak Ektachem DT60 analyzer (Rochester, New York) amylase content in plasma separation and measurement, using IU / L.
(3) the detection of MPO in lung tissue according to the kit instructions. The lung tissue homogenate after mechanical water bath, colorimetric, refer to the following formula: MPO (U / g) = (wet tube OD value of control group / OD) 11.3 x sample volume (g)
(4) to detect the expression of ICAM-1 protein in lung tissue of Western Blot method, gel imaging system on the photographic and determination of strip size and gray value, to strip the area * gray value expression represented by area /Actin band gray value X protein.
(5) to detect the relative activity of NF- K B: the density analysis using Leica Q500Mc image analysis system to the gray value of NF- kappa B relative activity changes.
3, to study the expression and significance of I / R MIF and MIP-2 in acute lung injury
(1) histopathological scores of rats in each group: take the head of the pancreas tissue and the lower lobe of the right lung tissue was fixed by 4% paraformaldehyde, embedded in paraffin, HE staining to observe the pathological changes and evaluated by light microscope.
(2) blood samples were collected after centrifugation, using Kodak Ektachem DT60 analyzer (Rochester, New York) amylase content in plasma separation and measurement, using IU / L.
(3) the detection of MPO in lung tissue according to the kit instructions. The lung tissue homogenate after mechanical water bath, colorimetric, refer to the following formula: MPO (U / g) = (wet tube OD value of control group / OD) 11.3 x sample volume (g)
(4) to detect the expression of MIF mRNA in lung tissue by RT-PCR, total RNA was extracted from lung tissue using Trizol method, RNA concentration was determined by UV spectrophotometer. The system analysis with the gray value of TC 21000 data to image analysis, gray level ratio MIF/GADPH, the relative expression of MIF mRNA values.
(5) to determine the content of MIP-2 in lung tissue: the lung tissue was homogenized with pre cooling medium 10 times the volume of the homogenate, a ELISA method was used to detect the concentration of MIP-2, according to the rMIP-2/GRO- beta ELISA kit provided by ELISA automatic tester instructions set a reaction step; by using automatic biochemical analyzer for the determination of protein content in animals.
4, statistical analysis
Statistical analysis was performed using SPSS 13 software. The test of normality of data, with the mean (Mean) and standard deviation (SD) described a normal distribution of data and discrete levels. All indexes between groups were compared by t test before and after the test, compared with paired t test, P value of 0.05 that have statistical significance.
Result
1, study on airway hyperresponsiveness induced by ischemia reperfusion of pancreas
This study found that nitric oxide in I / R mice blood, hydroxyl radical, amylase, tumor necrosis factor, significantly increased leukocyte concentration. In ischemia / reperfusion (I / R) expression of iNOS and tumor necrosis factor after mRNA in the pulmonary tissue increased significantly, lung function data show that the pancreas the ischemia / reperfusion (I / R) induced airway reactivity to methacholine increased; compared with sham operation group, I / R in the PenH group increased significantly, and lavage leucocyte increased significantly.
2, NF- kappa B and role of ICAM-1 in I / R complicated with lung injury
The pathological group of rat pancreas and lung tissue I / R scores were 5.94 + 0.72 and 6.42 + 0.65; significantly higher than the group Sham rats pathological score (respectively: 0.20 + 0.14 and 0.27 + 0.31) (P0.05); serum amylase levels in rats of Sham group is 1198.4 + 121.7 / I; the rats in group R serum amylase level was 3719.6 + 523.8; the difference between the two groups reached statistical significance (P0.05). Compared with Sham rats, the expression level of MPO increased significantly in the lung tissues of pancreatic ischemia-reperfusion (0.74 + 0.06) (P0.05); ICAM-1 protein and ICAM-1 mRNA weak expression the lung tissue in Sham rats, increased ICAM-1 protein and ICAM-1 mRNA expression in lung tissue of rats in group I / R, the expression levels were 0.47 + 0.03 and 1.12 + 0.07; pancreatic ischemia reperfusion injury can make NF- kappa B activity in lung tissue of rats increased significantly.
3, to study the expression and significance of I / R MIF and MIP-2 in acute lung injury
Pancreas and lung tissue in Sham group pathological score were 2.14 + 0.06 and 0.37 + 0.14; pathology of lung and pancreatic tissues of I / R scores were 8.52 + 1.17 and 4.71 + 0.30; difference between the pathological scores of the two groups reached statistical significance (P0.05). The serum expression of.Sham in pancreatic ischemia reperfusion rats the level of MPO increased significantly in serum amylase and lung tissue in MIF and lung tissue MIFmRNA expression was weak, while MIF, lung tissue and serum I / R rats the expression of MIFmRNA in.Sham group increased the level of MIP-2 activity in the lung tissues of rats was 23.9 + 5.8; pancreatic ischemia reperfusion injury can make the levels of MIP-2 activity in lung tissue of rats was significantly increased (91.5 + 12.1).
conclusion
In 1, pancreatic I / R induced systemic inflammatory response and lung leukocyte (WBC) increased. Then airway hyperresponsiveness in reperfusion group may be due to airway inflammation, which increases the expression of iNOS in lung tissue and lung gathered in WBC, tumor necrosis factor, expression of inflammatory mediators.
2, can increase ICAM-1 protein and ICAM-1 expression in lung ischemia reperfusion injury of pancreas. The level of NF- K B in lung tissue of pancreatic ischemia reperfusion for 2 hours after 6 hours.
3, can increase the MIF and MIFmRNA gene expression levels in serum and lung tissue of pancreatic ischemia reperfusion injury. The increased content of MIP-2 in lung tissue, as a pro-inflammatory cytokine early, MIP-2 mediated lung injury.

【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

【参考文献】