【作者】 李洁；

【导师】 戴爱国；

【作者基本信息】 南华大学 ， 内科学， 2010， 硕士

【摘要】 【目的】研究PPARγ、PGC-1α、Nrf2及γ-GCS-HS在慢性阻塞性肺疾病(COPD)大鼠及患者肺组织中的表达变化关系,探讨PPARγ及其激活剂罗格列酮(RGZ)、PGC-1α对Nrf2、γ-GCS-HS基因表达的影响及它们在COPD发病中的作用,为COPD的防治提供新的理论依据。【方法】分为动物实验和临床实验两部分。(1)动物实验:健康雄性SD大鼠36只,随机分为对照组、COPD模型组和RGZ干预组,每组12只。采用每日熏香烟和两次气管内滴入脂多糖(LPS)法制作大鼠COPD模型,同时利用PPARγ激活剂RGZ对其进行干预。测定大鼠肺功能并观察大鼠肺组织病理形态学改变;原位杂交和逆转录-聚合酶链反应(RT-PCR)检测大鼠肺组织中PPARγ、PGC-1α、Nrf2、γ-GCS–HS mRNA的表达。免疫组化和免疫印迹(western blot)分析大鼠肺组织中PPARγ、PGC-1α、Nrf2、γ-GCS-HS的蛋白表达水平。(2)临床实验:手术切除肺癌患者肺组织标本40例,所有患者术前均行肺功能检测,分为对照组、轻度COPD组、中度COPD组和重度COPD组,各COPD组患者均按照中华医学会2007年版COPD诊治指南进行严重程度分级,原位杂交分析各组患者肺组织中PPARγ、PGC-1α、Nrf2和γ-GCS-HS mRNA水平,免疫组织化学检测各组患者肺组织中PPARγ、PGC-1α、Nrf2和γ-GCS-HS蛋白的表达。【结果】1.动物实验结果:(1)大鼠肺功能指标(FEV0.3、FEV0.3/FVC%、PEF)在COPD组较对照组显著降低(P<0.01),RGZ干预组较对照组降低(P<0.01),较COPD组明显增高(P<0.01)。COPD组大鼠肺部病理改变符合COPD的形态学特征,而RGZ干预组大鼠肺组织病理改变较COPD模型组明显改善。(2)ROS含量在COPD组较对照组显著增高(P<0.01),在RGZ干预组较COPD组显著降低(P<0.01)。γ-GCS-HS活性在COPD组和RGZ干预组均较对照显著升高(均P<0.01),RGZ干预组较COPD组进一步增高(P<0.01)。(3)原位杂交结果显示PPARγ、PGC-1α、Nrf2和γ-GCS-HS mRNA在三组大鼠肺组织中的表达部位基本一致,主要见于肺泡、细支气管上皮细胞、支气管平滑肌细胞和部分血管平滑肌细胞。免疫组化结果显示PPARγ、PGC-1α、Nrf2、γ-GCS-HS蛋白主要表达于支气管、细支气管上皮细胞、肺泡上皮细胞,PPARγ、PGC-1α、Nrf2以胞核表达为主,γ-GCS-HS在胞浆胞核均有免疫着色。(4)大鼠肺组织中PPARγ、PGC-1α和γ-GCS-HS mRNA在COPD组和RGZ干预组均显著高于对照组(P<0.01),而在RGZ干预组明显高于COPD组(P<0.05);Nrf2 mRNA在COPD组和对照组均呈阳性表达,两组比较差异无统计学意义(P>0.05),而在RGZ干预组呈强阳性表达且较对照组和COPD组明显增高(P<0.01)。(5)大鼠肺组织中PPARγ、PGC-1α、Nrf2、γ-GCS-HS蛋白表达在COPD组和RGZ干预组均显著高于对照组(均P<0.01),而在RGZ干预组均较COPD组进一步增高(均P<0.05)。(6)SPSS13.0软件进行直线相关分析,在动物实验中PPARγ、PGC-1α蛋白表达与Nrf2蛋白及mRNA表达均呈正相关(均P<0.01),PGC-1α蛋白表达与ROS含量呈正相关(P<0.01),PPARγ、Nrf2蛋白表达与ROS含量无明显相关性(P>0.05)(Tab 8)。PPARγ、PGC-1α、Nrf2蛋白表达与γ-GCS-HS mRNA、蛋白表达、酶活性均呈正相关(均P<0.01)。2.临床实验结果(1)轻度COPD患者肺组织中PPARγ、PGC-1α和γ-GCS-HS mRNA呈强阳性表达,较对照组明显增高(均P<0.01),而在中度COPD和重度COPD患者肺组织中PPARγ、PGC-1α和γ-GCS-HS mRNA表达呈进行性下降,较对照组和轻度COPD组明显降低(均P<0.01)。Nrf2 mRNA在各组COPD患者和对照组的肺组织中均呈阳性表达,各组比较差异无统计学意义(P>0.05)。(2)PPARγ、PGC-1α、Nrf2和γ-GCS-HS蛋白在轻度COPD患者肺组织中呈强阳性表达,较对照组明显增高(均P<0.01),而在中度COPD和重度COPD患者肺组织中表达呈进行性下降,较对照组和轻度COPD组明显降低(均P<0.01)。(3)直线相关分析显示在临床实验中PPARγ、PGC-1α、Nrf2、γ-GCS-HS蛋白表达与FEV1(%)均呈正相关(均P<0.01),与FEV1/FVC(%)无明显相关性(P>0.05)。PPARγ、PGC-1α蛋白表达与Nrf2蛋白、γ-GCS-HS mRNA及蛋白表达均呈明显正相关(均P<0.01),而与Nrf2 mRNA表达无明显相关性(P>0.05)。【结论】1.在COPD早期阶段抗氧化酶γ-GCS可能代偿性上调以抵抗氧化应激,而随着COPD严重程度的加深γ-GCS逐渐失代偿呈进行性下调。2. PPARγ和PGC-1α通路活化可能减轻COPD氧化/抗氧化失衡,两者可能通过上调γ-GCS的酶活性和基因表达及减少ROS的含量而发挥抗氧化保护作用,参与COPD的进展过程,对COPD的防治具有重要意义。3.在COPD发病过程中,氧化应激可能主要诱导Nrf2的蛋白表达,而PPARγ和PGC-1α通路活化可能通过影响Nrf2 mRNA及蛋白表达水平而上调γ-GCS,从而增加GSH合成,对抗氧化应激。更多还原

【Abstract】 【Objective】To evaluate the expression patterns and their relationship of PPARγ、PGC-1α、Nrf2 andγ-GCS-HS in lungs of rats and patients with COPD,to investigate effects of PPARγand its agonist RGZ、PGC-1αon gene expression of Nrf2 andγ-GCS-HS, and its role in the pathophysiological course of chronic obstructive pulmonary disease,and provide a new theoretical basis for the prevention and treatment of COPD【Methods】The study was composed of two parts: animal experiment and clinical trial. (1)Thirty six adult male Sprague- Dawley(SD) rats were randomly divided into control group、COPD model and RGZ-treated group with twelve rats in each group. The rat COPD model was established by intratracheal instillation of lipopolysaccharide twice and exposed to cigarette smoke daily. The lung function measurements were carried out , and the pathological changes were observed. mRNAs expression of PPARγ、PGC-1α、Nrf2、γ-GCS-HS in lung tissue of rats was measured by in site hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR) . Proteins expression of PPARγ、PGC-1α、Nrf2、γ-GCS-HS were observed by immunohistochemistry(IH) and western blot.(2) Clinical peripheral lung tissue samples were obtained from forty patients undergoing resection for lung cancer, and all patients were were measured lung function prior to they were operated.Those patients were divided into four groups: controls, Mild COPD group、Moderate COPD group、Severe COPD group. COPD classification based on the Chinese Medical Association established standards in 2007. mRNA expression of PPARγ、PGC-1α、Nrf2、γ-GCS-HS in clinical lung tissue was measured by in site hybridization (ISH) . Protein expression of PPARγ、PGC-1α、Nrf2、γ-GCS-HS in clinical lung tissue were observed by immunohistochemistry(IH). 【Results】1. Animal experiments:(1)The results of rats pulmonary function showed: forced expiratory volume in first 0. 3 second(FEV0.3)、percentage of forced expiratory volume in first 0. 3 second to forced vital capacity (FEV0.3/FVC%) and peak expiratory flow (PEF) were all obviously decreased in COPD group compared with control group (P<0.01) and which were all significantly improved in RGZ-treated group compared with COPD model group (all P < 0.01).Lung pathological changes in COPD model group conformed morphological character of COPD,however, pathological changes of rats lung tissue in RGZ-treated group markedly reduced compared with COPD model group.(2)In COPD model group, ROS levels was markedly raised compared to controls(P<0.01), whereas in RGZ-treated group, ROS levels was remarkably reduced compared to COPD model group(P<0.01).γ-GCS-HS activity assay showed that, in COPD model group and RGZ-treated group,γ-GCS-HS activity was all observably increased compared to controls(all P<0.01), and which was further increased in RGZ-treated group compared to COPD model group(P<0.01).(3)In situ hybridization showed that the position of PPARγ、PGC-1α、Nrf2 andγ-GCS-HS mRNAs expression are basically consistent in the lung tissue of three group rats, which were mainly located in alveolar epithelial cells、bronchial epithelial cells、bronchial smooth muscle cells and part of the vascular smooth muscle cells. proteins expression of PPARγ、PGC-1α、Nrf2 was mainly found in the nucleus of alveolar epithelial cells and bronchial epithelial cells of rats in each group, while, staining ofγ-GCS-HS proteins was found not only in the nucleus but also in the cytoplasm of alveolar epithelial cells and bronchial epithelial cells of rats in each group(4)PPARγ、PGC-1αandγ-GCS-HS mRNAs in rat lung tissues were significantly higher in COPD model group than those in controls(P<0.01), and those were further elevated in RGZ-treated group than those in COPD model group(P<0.05). mRNA expression of Nrf2 were positive in COPD model group and control group,and there were no significant difference between the two groups(P>0.05),while mRNA expression of Nrf2 were strongly positive in RGZ-treated group,and there were significant differences compared with COPD model group and control group(P<0.01).(5)Proteins expression of PPARγ、PGC-1αandγ-GCS-HS in rat lung tissues were significantly higher in COPD model group and RGZ-treated group than those in controls(P<0.01), and those were manifestly increased in RGZ-treated group compared with COPD model group(P<0.05).(6)Linear correlation analysis showed that proteins expression of PPARγ、PGC-1αwere positively correlated with Nrf2 mRNA and protein(all P<0.01), proteins expression of PGC-1αwas positively correlated with ROS contents(P<0.01), and expression of PPARγ、Nrf2 were no significantly correlated with ROS contents(P>0.05). proteins expression of PPARγ、PGC-1α、Nrf2 were positively correlated withγ-GCS-HS activity、mRNA and protein (all P<0.01).2. Clinical experiments:(1)mRNAs expression of PPARγ、PGC-1α、γ-GCS-HS were strongly positive in lungs of patients with Mild COPD, and which were markedly increased compared with normal control subjects(all P<0.01), and showed a progressive decrease in lungs of patients with Moderate and Severe COPD,and were obviously decreased compared with normal control subjects and patients with Mild COPD(all P<0.01). mRNA expression of Nrf2 Showed weakly positive in lungs of each group patients with COPD and normal control subjects,and there are no significant differences between groups(P>0.05).(2)Proteins expression of PPARγ、PGC-1α、Nrf2、γ-GCS-HS were strongly positive in lungs of patients with Mild COPD, and which were markedly increased compared with normal control subjects(all P<0.01), and showed a progressive decrease in lungs of patients with Moderate and Severe COPD, and which were obviously decreased compared with normal control subjects and patients with Mild COPD(all P<0.01).(3)Linear correlation analysis showed proteins expression of PPARγ、PGC-1α、Nrf2、γ-GCS-HS were positively correlated with FEV1(%)(all P<0.01),were no significantly correlated with FEV1/FVC(%)(P>0.05). Proteins expression of PPARγ、PGC-1αwere positively correlated with Nrf2 protein、γ-GCS-HS mRNA and protein(all P<0.01), and were no significantly correlated with Nrf2 mRNA(P>0.05).【Conclusions】1. Antioxidant enzymeγ-GCS may be compensatory up-regulate to against oxidative stress in the early stage of COPD, but which maybe a progressive decompensation with increasing COPD severity.2. Activation of the PPARγand PGC-1αpathway may reduce the extent of COPD oxidant/antioxidant imbalance , and they both may protect against COPD progression by up-regulatingγ-GCS enzyme activity and gene expression as well as relieving ROS levels.3. In the pathogenesis of COPD, oxidative stess may mainly induce expression of Nrf2 protein,however,activation of the PPARγand PGC-1αpathway may up-regulatingγ-GCS to increase GSH synthesis against oxidative stress by influencing levels of Nrf2 mRNA and protein.更多还原