【作者】 冯青青；

【导师】 夏熙郑；

【作者基本信息】 郑州大学 ， 呼吸内科， 2013， 硕士

【摘要】 背景及目的慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)是呼吸系统疾病中比较常见的慢性疾病。该病患病人数多,病死率高,且呈进行性发展,到疾病的终末期严重影响了患者的劳动力,生活质量亦明显下降。目前COPD的发病机制尚未完全明了,但研究证实氧化应激是COPD重要的发病机制之一。核因子相关因子-2(Nuclear factor erythroid2-related factor2,Nrf2)是氧化应激反应中位于核心地位的转录因子,能够上调多种抗氧化基因的表达,在COPD的发病过程中发挥了重要的作用。BTB-CNC异体同源体1(BTB and CNC homolog1, Bachl)是广泛存在于体内的一种亚铁血红素结合蛋白,它与Nrf2不同,可以阻遏抗氧化基因的转录。还原型谷胱甘肽(GSH)是肺组织内重要的抗氧化剂之一,在氧化剂对肺组织造成氧化损伤的过程中发挥了重要的保护作用。在GSH的合成反应过程中,调节GSH合成速度和数量的重要限速酶是γ-谷氨酸半胱氨酸合成酶(y-Glutamylcysteine Synthetase,y-GCS)。γ-GCS表达的调节主要集中在转录水平,因此,我们可以在转录水平对γ-GCS的表达进行调节,从而来增加GSH的合成,提升肺部的抗氧化能力。在对COPD大鼠的研究中表明Nrf2、Bachl在大鼠COPD的发病过程中可能对抗氧化基因γ-GCS的表达起到竞争调节的作用。国外有研究发现Bachl可以与Nrf2竞争抗氧化反应原件结合位点,从而下调抗氧化基因的表达。本实验旨在对COPD临床患者肺组织中Nrf2、Bachl、γ-GCS的表达水平的变化及相关性进行研究,探讨三者在COPD发病机制中的作用,并初步探索Nrf2、Bachl对γ-GCS表达的调控作用。对象和方法选取2011年3月至2012年5月因肺肿瘤于郑州大学第二附属医院胸外科行肺叶切除术的患者40例。术前均询问病史,完善常规体检、拍摄胸部X线或CT、测肺功能等资料。凡符合中华医学会呼吸病学分会制定的慢性阻塞性肺疾病诊治指南者,入选COPD组(20例),不符合者则入选对照组(20例)。所有患者均排除合并患有心、肝、肾、皮肤、乳腺、肾脏、消化道、卵巢、血液等方面的疾病,并排除支气管扩张症、肺结核和其它可以影响肺功能的疾病,且术前均未行放化疗治疗。凡是符合入选标准的患者都已告知本课题的目的及方法,签署知情同意书,并给予患者适当的经济补偿。距离肿瘤组织4cm以上取材,肉眼未观察到肺癌浸润的外周肺组织,于4%多聚甲醛(含1‰焦炭酸二乙酯)中固定,常规行石蜡包埋,切片。取肺组织切片行HE染色；原位杂交法检测肺组织标本中Nrf2、Bach1、γ-GCSmRNA的表达；免疫组化法检测肺组织标本中Nrf2、Bach1、γ-GCS蛋白的表达；用SPSS17.0统计软件对实验数据进行统计学分析。结果1、临床患者肺组织切片HE染色结果：在光镜下可见COPD组肺泡壁变薄,肺泡腔变大,有的肺泡壁出现不同程度的断裂,同时可见有的断裂的肺泡壁融合。而对照组患者肺泡结构正常。2、原位杂交结果：Nrf2、Bachl和γ-GCS mRNA主要表达于肺泡上皮细胞和炎症细胞；Nrf2和Bachl mRNA在COPD组患者肺组织中均呈强阳性表达,二者在对照组的相应部位呈弱阳性表达,两组比较差异有统计学意义(P＜0.05)。而γ-GCS mRNA在COPD组患者肺组织中呈阳性表达,在对照组的相应部位呈弱阳性表达,两组比较差异亦有统计学意义(P＜0.05)。3、免疫组化结果：Nrf2、Bach1、γ-GCS蛋白主要表达在肺泡上皮细胞和炎症细胞。Nrf2、Bachl、γ-GCS蛋白在COPD组患者肺组织中均呈强阳性表达,与对照组相比较差异有统计学意义(P＜0.05)。4、COPD组指标间的相关性分析：Nrf2、γ-GCS蛋白与FEV1/FVC(%)、FEV1(%)均呈正相关(P<0.01),Bachl蛋白与FEV1/FVC(%)、FEV1(%)呈负相关(P＜0.01)。Nrf2蛋白与γ-GCS mRNA及其蛋白的表达呈正相关(P＜0.01),Bachl蛋白与γ-GCS mRNA及其蛋白的表达呈负相关(P＜0.01)。结论1、COPD患者肺组织中Nrf2、Bach1、γ-GCS mRNA及蛋白的表达均较对照组增强,提示三者在COPD氧化/抗氧化系统中发挥了重要作用。2、Nrf2、Bachl、γ-GCS蛋白与诊断及评估COPD的关键指标FEV1/FVC(%).FEV1(%)的相关性分析结果进一步证实三者可能与COPD的发病有着密切的关系。3、在COPD的发病过程中,Nrf2蛋白对γ-GCS的基因表达可能起到正向调控的作用,Bachl蛋白对γ-GCS的基因表达可能起到负向调控的作用,为γ-GCS的调控机制提供了新的理论依据。更多还原

【Abstract】 Background and ObjectiveChronic obstructive pulmonary disease (COPD) is a common and chronic disease of the respiratory system. Because of the high disease prevalence and mortality, the progressive development, this disease can seriously affect labor ability and the quality of life of patients at the end-stage. At present,the pathogenesis of COPD is unclear, but the studies have confirmed that oxidative stress is one of the important pathogenesis of COPD. Nuclear factor erythroid2-related factor2(Nrf2) is the nuclear transcription factor in oxidative stress reaction, which can positively regulate the expression of a variety of antioxidant genes,and plays an important role in the pathogenesis of COPD. BTB and CNC homologl Bachl (Bachl) is one of the heme binding proteins, which widely exists in the body.But it can inhibit the transcription of oxidation resistance genes,that is different from Nrf2.Reduced glutathione (GSH) is an important antioxidant,which plays an important role in the process of resisting the Oxidative damage of lung. y-Glutamylcysteine Synthetase (y-GCS) is an important enzymes,which can limit the speed of synthesis of GSH,also can decided the speed and quantity of the synthesis GSH.The expression of y-GCS is mainly regulated in the level of transcription, thus, we can increase the synthesis of GSH to enhance antioxidant capacity of lung through this way. The studies of COPD rats show that Nrf2can compete with Bachl to regulate the expression of the antioxidant gene of y-GCS in the process of the development of COPD.Some Overseas studies have found that Bachl can downgrade the expression of antioxidant gene through competing with Nrf2to bind the original oxidation reaction. This experiment aims to study the change of the expression level of Nrf2, Bachl, y-GCS in the lung tissue of COPD and the correlation between them, to explore their roles in the pathogenesis of COPD and how Nrf2and Bachl regulate the expression of γ-GCS.Subjects and methods40patients were selected from the thoracic surgery of the second affiliated hospital of zhengzhou university from March2011to May2012, who had done Lung resection surgery because of lung cancer. We asked the medical history of each person,done the regular check-up, chest X-ray or CT, lung function test and so on before the surgery.He would be included in the COPD group (20cases),who conformed to the diagnosis and treatment of COPD guideline set by the respiratory branch of Chinese Medical Association.If he didn’t conform to the guideline,would be in the control group (20cases). The patient was excluded suffering from heart, liver, kidney, skin, breast, kidney, digestive tract, ovary, blood diseases, ect. The patient was also eliminated suffering from bronchiectasis disease, tuberculosis and other diseases which could affect the function of pulmonary, and had not done preoperative chemoradiation therapy.All patients who met the inclusion criteria, were told that the purpose of this study and methods, signed informed consent form, and would be given appropriate economic compensation.the samples were taken from the tumor tissue more than4cm, and were the peripheral lung tissue not infiltrated by Lung cancer through macroscopic observation. The samples were fixed in4%paraformaldehyde (including1%oDiethypyrocarbonat), and sectioned them after conventional paraffin embedding. Then we done the HE staining;In situ hybridization and immunohistochemistry were used to detect the mRNA and protein expressions of Nrf2,Bachl and y-GCSmRNA in the lung tissues of two groups of patients. SPSS17.0statistical software was used for statistical analysis of experimental data.Results 1. Lung tissue pathological slices by HE staining under the optical microscope:In COPD group, alveolar walls become thinner, and appeared different degree of fracture, some parts of the fractured alveolar fused. The structure of alveolar was normal in the control group.2. In situ hybridization results:The expressions of Nrf2, Bachl and y-GCS mRNA were mainly in alveolar epithelial cells and inflammatory cells. In the lung tissue of patients in COPD group, Nrf2and Bachl mRNA were strong positively expressed, γ-GCSmRNA was positively expressed.Compared with the control group,the difference was statistically significant (P<0.05).3. Immunohistochemical results:The expressions of Nrf2, Bach1, γ-GCS proteins were mainly in alveolar epithelial cells and inflammatory cells.In the COPD group, Nrf2, Bach1, γ-GCS proteins were strong positively expressed.Compared with the control group, the difference was statistically significant (P<0.05).4. Correlation analysis in the COPD group:The expressions of Nrf2,y-GCS proteins and FEV1/FVC(%), FEV1(%) were positively correlated (P<0.01). The expression of Bachl protein and FEV1/FVC(%), FEV1(%) were negatively correlated (P<0.01). The expression of Nrf2protein and the expressions of y-GCS mRNA, γ-GCS protein were positively correlated (P<0.01), The expression of Bachl protein and the expressions of γ-GCS mRNA, γ-GCS protein were negatively correlated (P<0.01).Conclusions1.In the lung tissue of patients with COPD, the expressions of Nrf2, Bachl, y-GCS mRNA and its protein were enhanced.The result showed that all of them played an important role in the oxidation/antioxidation system of COPD.2. The correlation analysis results of Nrf2, Bachl, γ-GCS proteins and FEV1/FVC (%),FEV1(%),which were both the key indicators of diagnosis and evaluation of COPD, further confirmed that all of them may have a close relation to COPD.3.In the process of the development of COPD, Nrf2may positively regulate the expression of GCS gene, Bachl may negatively regulate the expression of GCS gene. The result provided a new theoretical basis of regulatory mechanism for γ-GCS.更多还原