【作者】 何白梅；

【导师】 罗百灵；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 研究背景肺泡上皮细胞凋亡是COPD发病的重要机制之一。内质网应激(ERS)介导的细胞凋亡途径是一条新的细胞凋亡途径。有关ERS标志性分子GRP78的生物学功能的研究已经引起生物学家们的广泛重视,GRP78在ERS时表达增加,被认为可以减轻ERS,从而减少细胞凋亡。GRP78表达上调的信号传导通路目前不完全清楚,有研究提示p38MAPK途径可以上调GRP78的表达。吸烟是COPD最重要的发病因素。香烟烟雾中富含氧自由基和多种毒性成分,氧化应激和毒性物质都是ERS的诱发因素,因此吸烟很可能触发ERS。国内外研究均证实,在COPD中,香烟烟雾可以引起p38MAPK的活化。结合上述研究,我们推测在COPD发生过程中,吸烟可能通过诱导ERS导致肺泡上皮细胞凋亡从而参与COPD肺气肿的形成,在此过程中GRP78表达上调,发挥抗凋亡作用,p38MAPK途径在香烟烟雾诱导肺泡上皮细胞GRP78表达过程中起重要的调控作用。本研究拟以COPD大鼠及香烟烟雾提取物(CSE)干预的A549细胞为主要模型,探讨香烟烟雾诱导ERS的发生、GRP78的抗凋亡作用以及p38MAPK途径在GRP78表达过程中的调控作用。第一章COPD大鼠肺组织内质网应激与细胞凋亡目的观察COPD大鼠肺组织内质网应激的发生及肺泡上皮细胞的凋亡情况。方法24只Wistar大鼠随机均分为2组：正常对照组和COPD模型组。采用被动吸烟加气管内注射脂多糖法建立COPD大鼠模型。造模完成后,测定各组大鼠的肺功能、观察肺组织病理学变化、免疫组化检测GRP78在肺组织中的表达和分布,RT-PCR检测GRP78、CHOP mRNA水平,Western blot检测GRP78、CHOP、active caspase-12蛋白水平。TUNEL法检测肺泡上皮细胞凋亡结果COPD模型组大鼠FEV0.3/FVC (%)、动态肺顺应性(Cdyn)均较对照组明显降低,而气道阻力(RI)明显增高；免疫组化结果显示GRP78表达于肺泡上皮细胞、支气管上皮细胞、血管内皮细胞等细胞的细胞浆中,以肺泡上皮细胞明显。对照组GRP78呈弱阳性表达,阳性产物为棕黄色颗粒；模型组GRP78呈强阳性表达,阳性产物为棕褐色颗粒。与对照组相比,模型组肺组织GRP78表达显著增高；RT-PCR结果显示COPD模型组大鼠支气管肺组织中GRP78 mRNA和CHOP mRNA表达量较对照组显著增高；Western blot结果显示COPD模型组大鼠肺组织中GRP78、CHOP、active caspase-12蛋白表达较正常对照组显著增高；TUNEL法结果显示COPD模型组肺泡上皮细胞凋亡指数较正常对照组显著增高。结论采用被动吸烟加气管内注射脂多糖的方法,成功复制出了COPD大鼠模型;COPD大鼠肺组织发生了内质网应激,尤以肺泡上皮细胞明显；COPD大鼠肺泡上皮细胞凋亡增加,内质网应激可能是介导肺泡上皮细胞发生凋亡的重要途径。第二章香烟烟雾提取物诱导肺泡上皮细胞GRP78的表达及其抗凋亡作用目的观察CSE对A549细胞GRP78表达的影响,并探讨GRP78抗细胞凋亡作用。方法第一部分,体外培养A549细胞,给予不同浓度(0%-10%)和不同时间(0h-24h)的CSE干预后,以RT-PCR、Western blot分别检测GRP78 mRNA和蛋白表达；第二部分,将A549细胞分为四个处理组：对照组,5%CSE组,GRP78siRNA+5%CSE组,control siRNA+5%CSE组。以RT-PCR、Western blot分别检测GRP78 mRNA和蛋白的表达水平,Western blot检测active caspase-3蛋白表达水平,TUNEL检测A549细胞凋亡指数(AI)的变化。结果随着CSE浓度的增加或干预时间的延长,A549细胞GRP78 mRNA及GRP78蛋白表达量逐渐增加,以5%CSE、干预12h组增加最明显,但继续增加CSE浓度或延长干预时间,GRP78表达并不增加,反而下降；小干扰GRP78后,RT-PCR、Western blot检测小干扰组GRP78表达显著下降,GRP78siRNA成功下调了GRP78基因表达；GRP78水平下调后,A549细胞active caspase-3的蛋白表达水平和细胞凋亡指数与单纯的CSE刺激组比较显著升高。结论低浓度、短时间CSE刺激后,A549细胞启动了保护性的非折叠蛋白反应,GRP78表达上调。但高浓度、长时间CSE刺激,内质网出现功能障碍,其保护机制失代偿,GRP78表达反而下降；GRP78抵抗香烟烟雾提取物诱导的A549细胞凋亡。第三章p38MAPK在香烟烟雾提取物诱导肺泡上皮细胞GRP78表达过程中的调控目的明确p38MAPK在CSE诱导肺泡上皮细胞GRP78表达过程中的调控作用。方法第一部分,24只Wistar大鼠随机均分为2组：正常对照组和COPD模型组。采用被动吸烟加气管内注射脂多糖法建立大鼠COPD模型。造模完成后,Western blot检测各组大鼠肺组织内P-p38、GRP78蛋白表达水平,并将二者进行相关性分析；第二部分,将A549细胞分为三个处理组：对照组,5%CSE组,SB203580+5%CSE组,以RT-PCR、Western blot分别检测GRP78 mRNA和蛋白的表达水平变化。结果COPD模型组大鼠支气管肺组织中P-p38蛋白表达水平较对照组显著升高,且P-p38蛋白水平与GRP78蛋白表达呈正相关(r=0.848, P<0.01); p38MAPK抑制剂SB203580干预后,P-p38蛋白表达水平显著降低。抑制了p38MAPK通路活化后再给予CSE干预,GRP78 mRNA及GRP78蛋白表达水平较单纯的CSE刺激组显著降低。结论COPD中p38MAPK途径发生了磷酸化激活；p38MAPK途径能上调香烟烟雾提取物诱导的GRP78表达水平。更多还原

【Abstract】 Background The apoptosis of alveolar epithelial cell plays an important role in the pathogenesis of chronic obstructive pulmonary disease(COPD). In recent years, it has been reported that endoplasmic reticulum(ER) may be a new location which participates in intracellular pathway of cell apoptosis. That is called ER stress-mediated apoptosis pathway. GRP78, a 78kDa glucose regulated protein, is a major ER chaperone and plays a critical role in regulating ER homeostasis. Previous studies revealed that p38 mitogen-activated protein kinase (MAPK) may be involved in the induction of GRP78. Cigarette smoke, has been regarded as the main risk factor that triggers development of COPD. Some components in cigarette smoke including ROS, nicotine, heavy metals, and aldehyde may have the potential to induce ER stress. It has been reported that oxidative stress can activate p38MAPK pathway, which play an important role in the inflammation of airway in COPD. Based on these results, we speculate that cigarette smoke can cause ER stress and thereby contributes to apoptosis of lung epithelial cells. Induction of GRP78 protects cells from apoptosis, and p38 MAPK may involved in the up-regulating of GRP78. In present study, we use smoke-induced COPD rat and cigarette smoke extract (CSE) treated human alveolar epithelial cells (A549) as models, to explore the mechanisms of ER stress in COPD, the anti-apoptosis role of GRP78 and the regulative effect of MAPK pathway in this process.Chapter One The ER stress and ER stress-mediated apoptosis in the lung tissues of COPD ratObjective To study the ER stress and the apoptosis of alveolar epithelial cell in COPD rat model.Methods 24 Wister rats were divided into two groups at random: control group and COPD group. COPD rat model was established by intratracheal instillation of lipopolysaccharide (LPS) twice and exposure to cigarette smoke daily. The spirometry was conducted and the pathological changes were observed after the model was established. The mRNA of GRP78 and CHOP were detected by reverse transcription-polymerase chain reaction (RT-PCR).The expression of GRP78 in the lung tissues was examined by immunohistochemistry and the protein expression of GRP78, CHOP and caspase-12 was detected by Western blot.TUNEL was used to analyze alveolar epithelial cell apoptosis.Results Significant decrease of FEV0.3/FVC, Cdyn and increase of RI were found in the COPD group compared with the control group. Immunoperoxidase staining showed GRP78 was localized in the cytoplasm of bronchial epithelial cells, endothelial cells, especially in alveolar epithelial cells, and the GRP78 expression significantly increased in the COPD group. The levels of GRP78 and CHOP mRNA were higher in the COPD group than in the control group. The expression of GRP78, CHOP and active caspase-12 protein were increased in the COPD group. Apoptosis was observed with an in situ TUNEL assay, more apoptotic alveolar epithelial cells were found in the COPD group.Conclusion The COPD rat models was successfully established by intratracheal instillation of LPS and exposure to cigarette smoke; ER stress was triggered in the lung tissues of COPD rat. Alveolar epithelial cell apoptosis was increased in the COPD group. The ER stress-mediated apoptosis pathway may participate in the alveolar epithelial cell apoptosis in COPD.Chapter Two GRP78 induction by cigarette smoke extract and its anti-apoptosis effectObjective To investigate the induction of GRP78 mediated by cigarette smoke extract(CSE), and the anti-apoptosis effect of GRP78.Methods In part one, cultured A549 cells were exposed to CSE at various concentrations and for defferent durations. GRP78 expression was detected by RT-PCR and Western blot. In part two, A549 cells were divided into 4 groups:control group,5%CSE group, GRP78siRNA+5% CSE group, control siRNA+5% CSE group, measured the GRP78 expression by RT-PCR and Western blot, detected the active caspase-3 expression by Western blot,and TUNEL assay was used to detect apoptosis.Results Part one, the expression of GRP78 was gradually increased as raising the CSE concentration or prolonging the treated time. When A549 cells were treated with 5% CSE for 12 hours, the GRP78 expression level reach the peak value. When continue to raise the CSE concentration or prolong the durations, the GRP78 expression was decreased. Part two, pretreated with GRP78 siRNA and then stimulated with CSE significantly reducing the GRP78 expression, indicated that GRP78 gene expression was successfully inhibited by GRP78 siRNA, accompany with the increase of active caspase-3 expression and A549 cells apoptosis.Conclusion In A549 cells, low CSE concentration or short stimulation time up-regulate GRP78 expression via unfolded protein response. But high CSE concentration or long duration could down-regulate GRP78 expression because of the broken compensation of protection mechanism; GRP78 counteract A549 cells apoptosis induced by cigarette smoke.Chapter Three CSE up-regulates GRP78 expression in A549 cells via p38MAPK pathwayObjective To investigate the regulative effect of p38MAPK pathway in GRP78 expression induced by CSE.Methods In part one,24 Wister rats were divided into two groups at random:control group and COPD group. COPD rat model was established by intratracheal instillation of LPS twice and exposure to cigarette smoke daily. After the model was established, P-p38 protein expression was detected by Western blot. The correlation analysis was used to evaluate the association between P-p38 protein level and GRP78 expression. In part two, A549 cells were divided into 3 groups:control group,5%CSE group, SB203580+5%CSE group. P-p38 expression was measured by Western blot, and GRP78 expression was detected by RT-PCR and western blot.Results Part one, the expression of P-p38 was significantly increased in the COPD group compared with the control group, and the correlation analyses showed that P-p38 protein level was positively correlated with GRP78 expression (r=0.848, P<0.01). Part two, P-p38 expression was significantly decreased in SB203580 pretreated group, demonstrated that p38MAPK pathway was successfully blocked by SB203580. The expression of GRP78 which was induced by CSE was inhibited by SB203580.Conclusion p38MAPK pathway was activated in COPD; CSE up-regulate GRP78,which was attributed to p38 pathway.更多还原