【作者】 许霞；

【导师】 肖伟；

【作者基本信息】 山东大学 ， 呼吸内科， 2009， 博士

【摘要】 目的研究香烟烟雾提取物（CSE）及细菌成分脂多糖（LPS）刺激肺泡上皮细胞PAI-1表达在COPD炎症过程中炎症因子网络形成和炎症细胞趋化中的作用。慢性阻塞性肺疾病（COPD）是一种慢性气道炎症性疾病,以不完全可逆的气流受限为特征,气流受限呈进行性发展,是气道对有害气体或有害颗粒（特别是吸烟）的异常炎症反应。纤溶酶原激活物抑制剂-1（PAI-1）是尿激酶型纤溶酶原激活物（uPA）的主要抑制剂,除了其经典的纤溶功能以外,还可以调节细胞黏附、运动和细胞因子水平,在机体免疫和炎症及感染的控制方面有着重要作用。PAI-1可以调节细胞迁移,但其在细胞迁移中的作用是有争议的,在不同的研究中,PAI-1显示了抑制和促进细胞迁移两种似乎矛盾的作用。PAI-1阻止ECM降解,抑制uPAR依赖的或整合素依赖的细胞黏附,阻断尿激酶型纤溶酶原激活物（uPA）诱导的趋化。通过这些作用,PAI-1抑制细胞迁移。PAI-1通过促进细胞分离,调节趋化物质,及其本身具有的趋化作用来促进细胞迁移。在体内实验中,PAI-1还能够调节某些细胞因子的水平。已证实PAI-1可以通过调节细胞因子的分布来影响细胞因子的水平,它能否直接影响细胞因子的表达有待进一步研究。前期研究显示,PAI-1在COPD患者的诱导痰中表达明显升高,且和肺功能重要指标FEV₁%负相关,与重要炎症因子IL-8水平正相关。这提示PAI-1在COPD的炎症过程中扮演着重要角色,因此我们进行本研究来明确PAI-1在COPD炎症中的作用。方法1.培养肺泡上皮细胞（A549细胞株）。2.根据Carp H.等的方法制备CSE。使用不同浓度的CSE和LPS刺激肺泡上皮细胞,ELISA检测细胞上清中PAI-1表达情况,找出PAI-1表达高的最佳刺激浓度进行下续实验。3.用RT-PCR检测CSE或LPS刺激肺泡上皮细胞后PAI-1mRNA表达的变化,用western blotting及免疫细胞化学法检测PAI-1蛋白表达。肺泡上皮细胞上清中的PAI-1及炎症因子IL-8,LTB4的表达使用ELISA检测。分离人静脉血中的单核细胞和中性粒细胞,进行checkerboard实验和Transwell实验,检测CSE和LPS刺激的肺泡上皮细胞趋化炎症细胞的情况。4.分别使用特异的针对PAI-1的3对siRNA转染肺泡上皮细胞,RT-PCR检验干扰效率,选择出干扰效率最高的一对siRNA。5.PAI-1干扰实验分为以下6组:ncRNA空白组,ncRNA+CSE刺激组,ncRNA+LPS刺激组,siRNA空白组,siRNA+CSE刺激组,siRNA+LPS刺激组。使用转染效率最高的PAI-1特异的siRNA或阴性对照ncRNA转染肺泡上皮细胞后,CSE或LPS刺激,使用RT-PCR,western blotting,ELISA,免疫细胞化学法检测PAI-1的表达,ELISA检测IL-8,LTB4的表达。Transwell小室法检测特异性干扰肺泡上皮细胞PAI-1表达后炎症细胞向其趋化的情况。6.用SPSS11.0软件进行统计学分析,两组之间比较采用成组设计资料两样本均数的t检验。结果1.ELISA结果显示暴露于10%CSE与75μg/mlLPS48小时后,PAI-1表达显著升高,且与其他浓度的CSE或LPS刺激产生的PAI-1有显著差异。2.RT-PCR结果显示,暴露于10%CSE或75μg/mlLPS的肺泡上皮细胞PAI-1mRNA表达明显增加;western blotting显示,暴露于10%CSE或75μg/ml LPS的肺泡上皮细胞PAI-1蛋白表达明显增加;免疫细胞化学结果显示,肺泡上皮细胞在受到10%CSE或75μg/ml LPS刺激后,细胞形态与对照组基本一致,而PAI-1呈显著增强的阳性表达;ELISA结果显示细胞上清中PAI-1表达与对照组相比也显著增加（P＜0.05,P＜0.01）,IL-8表达显著增加（P＜0.05,P＜0.01）,LTB4表达显著增加表达（P均＜0.01）。Transwell趋化实验结果显示单核细胞和中性粒细胞的趋化明显加强,10%CSE刺激后分别是对照组的2.8±0.1倍（P＜0.01）及2.45±0.12倍（P＜0.01）,75μg/ml LPS刺激后分别是对照组的2.25±0.11倍（P＜0.01）及2.9±0.15倍（P＜0.01）。3.RT-PCR结果显示,第三对PAI-1特异的siRNA具有最佳的干扰效率,PAI-1mRNA表达水平显著降低。4.免疫细胞化学结果显示,转染ncRNA和siRNA的肺泡上皮细胞形态基本一致,ncRNA+CSE刺激组和ncRNA+LPS刺激组可见肺泡上皮细胞与ncRNA空白组相比有增强的阳性表达,而siRNA空白组,siRNA+CSE刺激组和siRNA+LPS刺激组未见明显的阳性表达。RT-PCR和western blotting结果显示,通过使用PAI-1特异的siRNA,肺泡上皮细胞的PAI-1mRNA及蛋白表达都明显抑制,在受到CSE和LPS刺激后,也未见明显增加。ELISA结果显示,上清中IL-8表达siRNA空白组明显少于ncRNA空白组（P＜0.01）,siRNA+CSE刺激组IL-8表达与ncRNA+CSE刺激组相比较,明显减少（P＜0.05）。siRNA+CSE刺激组上清中LTB4表达显著低于ncRNA+CSE刺激组（P＜0.01）,siRNA+LPS刺激组上清中LTB4表达显著低于ncRNA+LPS刺激组（P＜0.01）。单核细胞和中性粒细胞的趋化在特异性干扰PAI-1表达后明显减弱。单核细胞趋化siRAN+CSE刺激组与ncRNA+CSE刺激组相比,siRAN+LPS刺激组与ncRNA+LPS刺激组相比,均明显减弱（P均＜0.01）。中性粒细胞趋化siRAN+CSE刺激组与ncRNA+CSE刺激组相比,siRAN+LPS刺激组与ncRNA+LPS刺激组相比,也同样显著减弱（P均＜0.01）。结论1.通过暴露于CSE和LPS,肺泡上皮细胞PAI-1、炎症因子（IL-8与LTB4）表达被明显诱导,炎症细胞（单核细胞和中性粒细胞）被趋化。2.PAI-1是调节CSE和LPS刺激的肺泡上皮细胞IL-8和LTB4表达的上游促炎症因子。3.PAI-1能够促进CSE和LPS刺激的肺泡上皮细胞对炎症细胞的趋化。4.干扰PAI-1表达可下调炎症因子表达,减少炎症细胞募集,进而减轻炎症反应。更多还原

【Abstract】 Background Chronic obstructive pulmonary disease is characterized by airflow limitation that is not fully reversible.The airflow limitation is usually progressive and associated with an abnormal inflammatory respose of the lung to noxious particles or gases.Chronic obstructive pulmonary disease is the fourth leading cause of death worldwide.It is mainly caused by exposure to cigarettes smoke,environmental or occupational pollutants,chronic infection or an interaction of these factors.It involves two major processes simultaneously,chronic airway inflammation and remodeling.The disbalance of proteinase and anti-proteinase is presumed important pathogenesis.Matrix metalloproteinase system(MMPs) plays an important role in the pathogenesie of COPD.Plasminogen activator inhibitor-1(PAI-1) can inhibit the activation of plasminogen to plasmin through urokinase type plasminogen activator (uPA) directly and MMPs indirectly.PAI-1 can regulate extracellular matrix (ECM) degradation,cell migration and levels of inflammatory factors.In this research,we study the effect of PAI-1 on the process of inflammation in COPD through exposuring alveolar epithelial cells(AECs) to cigarette smoke extraction(CSE) or lipopolysaccharides(LPS) and silencing the expression of PAI-1.Method CSE was prepared according to the method described by Carp H..Alveolar epithelial cells(A549 cells,AECs) were cultured and stimulated by different concentration of CSE or LPS.The levels of PAI-1 in supernatants were detected by ELISA.The concentration of CSE and LPS which induced the most PAI-1 expression was chosen and used in the next experiments.The mRNA and protein expression of PAI-1 in AECs were detected by RT-PCR and western blotting.The levels of PAI-1,interleukin-8(IL-8),leukotriene B4 (LTB4) in the supernatants of AECs were evaluated quantitatively by ELISA. Monocytes and neutrophils were separated from normal human veinous blood samples by Lymphoprep.Transwells were used to assess migration of monocytes and neutrophils to AECs.The AECs were transfected by siRNA that specially targeted PAI-1 or negative control RNA(ncRNA).RT-PCR was used to analysis the efficiency of transfection.After transfection,the AECs were exposured to CSE or LPS.The mRNA and protein expression of PAI-1 in AECs was evaluated by RT-PCR and western blotting.The levels of PAI-1, IL-8 and LTB4 in the supernatants of AECs were evaluated by ELISA. Transwells were used to assess migration of monocytes and neutrophils to AECs transfected by siRNA targeted PAI-1 or ncRNA and stimulated by CSE or LPS.Results The 10%CSE and 75μg/ml LPS can induce the most expression of PAI-1.The exposure to 10%CSE or 75μg/ml LPS enhanced expression of PAI-1,IL-8,LTB4 significantly(P＜0.05)and migration of monocytes and neutrophils to AECs(2.8±0.1fold,P＜0.01,2.45±0.12 fold, P＜0.01,2.25±0.11fold,P＜0.01 and 2.9±0.15 fold,P＜0.01,respectively). Through AECs transfection with siRNA targeted PAI-1,the expression of PAI-1 was inhibited significantly.Through silencing the expression of PAI-1, IL-8 and LTB4 induced by 10%CSE or 75μg/ml LPS were significantly attenuated compared to those without silencing the expression of PAI-1 under the same stimulation conditions(P＜0.05,P＜0.01,P＜0.01 and P＜0.01, respectively).The migration of inflammatory cells was attenuated significantly compared to those transfected by ncRNA under the same stimulation conditions(P＜0.01).Conclusion Through stimulation by CSE or LPS,the inflammation was induced in AECs,the expression of inflammatory factors(IL-8 and LTB4) was induced and inflammatory cells(monocytes and neutrophils) were recruited.PAI-1 promotes inflammation process induced by CSE or LPS in AECs through regulating expression of some inflammatory factors and chemotaxis of inflammatory cells.PAI-1 may play a proinflammatory role in the inflammation of AECs and the inflammation may be attenuated through silencing its expression. Inhibiting the expression of PAI-1 could be considered as a novel therapeutic strategy in COPD.更多还原