尿激酶型纤溶酶原激活物系统成分在COPD病理改变中作用的研究
发布时间：2019-05-28 21:12
【摘要】：研究背景 慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)是一种危害人类健康的常见病和多发病,其致残率、死亡率高,经济和社会负担重,严重降低病人和其家庭的生活质量,是每一个社会和政府必须面对并要解决好的公共卫生问题。目前,人们对于COPD本质的认识还远远不够,特别是对其主要的病理改变的原因和机制的了解还相当肤浅,造成COPD的诊断和防治上的不尽人意。 早在1957年就有学者提出,COPD的病变起始于小气道,随后的研究不断证实小气道病变在COPD发病中的重要地位。COPD病理改变最引人深思的是小气道壁胶原沉积和肺气肿的组织破坏的这种增生与降解共存的现象。研究和理解这种截然不同的病理结局,对于阐明发病机制,建立科学高效的诊治方法具有重要意义。 晚近的研究提示,细胞外基质合成/降解基因表达模式不同是造成COPD气道区和肺泡区产生截然不同病理改变的原因之一；与此同时,细胞过度凋亡在肺气肿形成中的作用也日益引起人们的关注。细胞外基质合成/降解和细胞凋亡/增殖的平衡失调可能共同参与了COPD的病理过程。对于COPD患者,其机体或肺组织中的某些系统在环境或致病因素的长期作用下,发生了异常变化,而引发COPD的病理改变。我们的前期研究不断证实了尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator, uPA)系统成分在COPD的炎症、组织重塑中有重要作用。尿激酶型纤溶酶原激活物系统,除了纤溶功能外,更具有非纤溶的多种功能,诸如调节细胞粘附、分化、运动以及凋亡/增殖等,参与多种疾病病理调控过程。本研究试图探究尿激酶型纤溶酶原激活物系统成分在COPD小气道胶原沉积与肺气肿形成中的作用,从一个新的角度阐释COPD的病理改变及其机制,为COPD的治疗提供新的靶点。 目的 探讨uPA系统在COPD病理改变中的作用及其相关机制： 1.观察COPD患者肺组织中uPA系统的分子表达模式,并分析其与COPD病理改变的关系； 2.明确uPA系统成分与COPD患者肺组织细胞凋亡的关系； 3.阐明uPA系统的主要成分之一尿激酶型纤溶酶原激活物抑制剂(PAI)-1在COPD肺泡上皮细胞凋亡中的作用及机制。 方法 1.病例选择 选择因良性病变行肺叶切除术的患者36例,术前行肺功能及CT检查,按照慢性阻塞性肺疾病全球倡议(GOLD)的诊断标准,将患者分为COPD组16例,肺功能正常对照组20例(其中吸烟对照组10例,不吸烟对照组10例)。 2.市组织标本的检测 (1)免疫组织化学方法检测肺组织标本uPA、尿激酶型纤溶酶原激活物受体(uPAR)、尿激酶型纤溶酶原抑制剂(PAI-1)、PAI-2的表达。 (2)天狼猩红染色检测小气道胶原沉积面积,分析肺组织中uPA系统成分表达与小气道壁胶原沉积的相关性；结合患者术前胸部CT资料,分析肺组织中uPA系统成分表达与肺气肿指数的相关性。 (3)原位末端转移酶标记技术(TUNEL)与免疫组织化学cleaved PARP染色,检测肺组织细胞凋亡情况,并分析uPA系统成分在肺组织表达与肺组织细胞凋亡的相关性。 3.体外细胞实验 (1)培养永生化人Ⅱ型肺泡上皮A549细胞系,应用脂质体2000将PAI-1小干扰RNA (siRNA)或无意义siRNA转染到细胞中。 (2) Real-time PCR、western blot和ELISA方法检测转染前后PAI-1表达,筛选出高效率抑制PAI-1表达的siRNA链。 (3)MTT方法检测CSC对A549细胞存活率的影响,选择合适浓度的CSC对转染后细胞进行刺激,模拟COPD细胞损伤过程。 (4) Annexin-Ⅴ和PI双染流式细胞技术检测细胞凋亡情况,探讨PAI-1对肺泡上皮细胞凋亡的影响。 (5)化学发光法检测转染前后细胞Caspase3/7、Caspase8以及Caspase9的活性,探讨PAI-1影响肺泡上皮细胞凋亡的通路。 (6)用Fas特异性中和抗体ZB4后重复转染实验及凋亡检测,进一步确定PAI-1影响肺泡上皮细胞凋亡的作用通路。 结果 1.肺组织标本检测 (1)uPA系统各成分在小气道区主要表达在小气道上皮细胞,其中,COPD组uPA、uPAR与PAI-2在小气道上皮细胞的表达均明显高于对照组(P0.05)；而PAI-1在COPD组小气道上皮细胞的表达低于对照组(P0.05)。在肺泡区,uPA系统成分主要表达在肺泡上皮细胞和肺泡巨噬细胞,其中COPD组uPA、uPAR在肺泡上皮和肺泡巨噬细胞的表达均显著高于对照组(P0.05),PAI-1在COPD组肺泡上皮细胞表达明显低于对照组,而在COPD组与对照组肺泡巨噬细胞中的表达无显著差异；PAI-2在COPD组与对照组肺泡上皮细胞与肺泡巨噬细胞表达均无显著差异。 (2) COPD患者小气道上皮uPA表达与气道壁胶原沉积面积呈正相关(r=0.4187,P=0.0011),且肺泡上皮细胞uPA的表达与肺气肿指数呈正相关(r=0.5188,p=0.0359)；COPD患者小气道上皮细胞PAI-2的表达与气道下胶原沉积面积呈正相关(r=0.5653,p=0.0225)。 (3) COPD组小气道区凋亡指数(AI)较对照组无显著差异,而肺泡上皮细胞AI较对照组显著增高(P0.05)；COPD患者肺泡上皮PAI-1的表达与肺泡上皮AI呈负相关(r=-0.5122,P0.05)。 2.体外细胞实验 (1) Real-time PCR、western bolt以及ELISA结果表明PAI-1siRNA可以显著抑制PAI-1表达,抑制率在90%以上。 (2)A549细胞存活率随CSC浓度增加及刺激时间延长而降低,具有浓度-时间-效应关系。 (3) siRNA干扰A549细胞PAI-1表达后,细胞凋亡增多(P0.05)。加入5%CSC刺激后,相比单纯干扰PAI-1,细胞凋亡也增多(P0.05)。 (4)干扰A549细胞PAI-1表达后,caspase3/7、caspase-8活性上调(P0.05),而caspase-9活性无明显变化(P0.05)。 (5)给予Fas中和抗体(ZB4)后,干扰PAI-1细胞与无意义干扰细胞凋亡水平的差异消失,而给予非特异性IgG,干扰PAI-1细胞凋亡水平仍显著高于无意义干扰细胞。 结论 1.uPA系统成分是参与COPD病理改变的重要物质。 2.肺泡上皮细胞PAI-1低表达很可能是引起肺泡上皮细胞过度凋亡的原因之一。 3.PAI-1可以抑制肺泡上皮细胞的过度凋亡,其作用是通过Fas-FasL通路实现的。
[Abstract]:Study Background Chronic obstructive pulmonary disease (COPD) is a common and multiple disease that is harmful to human health. It has a high rate of disability, high mortality, and heavy economic and social burden, which seriously reduces the quality of life of patients and their families. The quantity is a public health question that every society and government must face and solve At present, people's understanding of the nature of COPD is not enough, especially for the causes and mechanisms of the main pathological changes, and the understanding of the mechanism is rather superficial, leading to the diagnosis and prevention of COPD. In 1957, a scholar suggested that the pathological changes of COPD started with the small airway, and the subsequent study constantly confirmed the weight of the small airway disease in the pathogenesis of COPD. To be in position. The most thought-provoking of the pathological change in COPD is the coexistence of such hyperplasia and degradation of the tissue destruction of small-wall collagen deposition and emphysema The study and understanding of this distinct pathological outcome is of great significance for elucidating the pathogenesis and establishing a scientific and efficient diagnosis and treatment method. It is of great significance that the expression pattern of the extracellular matrix in the synthesis/ degradation of the extracellular matrix is one of the causes of different pathological changes in the airway region and the alveolar region of the COPD, and at the same time, the role of the apoptosis in the formation of the emphysema is also increasing. Concern is expressed that the balance of extracellular matrix synthesis/ degradation and cell apoptosis/ proliferation may be co-involved in COPD For COPD patients, some of the systems in their body or lung tissue have an abnormal change in the long-term effects of environmental or pathogenic factors, leading to COPD The pathological changes of urokinase-type plasminogen activator (uPA) in the inflammation and tissue remodeling of the patients with COPD have been confirmed in our early studies. in addition to that fibrinolytic function, the urokinase-type plasminogen activator system has various functions of non-fibrinolysis, such as regulation of cell adhesion, differentiation, movement, apoptosis/ proliferation, and the like, and is involved in various diseases The purpose of this study is to explore the role of urokinase-type plasminogen activator system in the formation of small airway collagen in COPD and the formation of emphysema, and to explain the pathological changes and mechanism of the COPD from a new point of view. supply Objective To study the target of uPA system in the pathological changes of COPD. 1. To observe the molecular expression pattern of the uPA system in the lung tissue of patients with COPD and to analyze the molecular expression pattern of the uPA system in the lung tissue of patients with COPD. the relationship between the pathological changes of COPD;2. Clear the component and COP of uPA system 3. The relationship between the apoptosis of lung tissue cells in patients with D.3. The urokinase-type plasminogen activator inhibitor (PAI)-1, one of the main components of the uPA system, was expressed in COPD. alveoli epithelial cell apoptosis Methods 1. The cases were selected to select 36 patients with pulmonary lobectomy for benign lesions, and the lung function and CT were performed in accordance with the Global Initiative for Chronic Obstructive Pulmonary Disease (GOL). D) The diagnosis standard of the patients was divided into 16 cases of COPD group and 20 cases of normal control group (n = 20). The smoking control group (10 cases) Non-smoking control group (n = 10).2. The detection of tissue specimen in the city (1) the detection of the tissue specimen of the city (1) the detection of uPA, urokinase-type plasminogen activator receptor (uPAR), and urinary shock in the lung tissue. Expression of the enzyme-type plasminogen activator (PAI-1) and PAI-2. (2) The relationship between the expression of uPA system components in the lung tissue and the collagen deposition of the small airway wall was analyzed by the staining of the tissue plasminogen activator (PAI-1) and the tissue plasminogen activator (PAI-1). The relationship between the expression of uPA system and the index of emphysema in lung tissue was analyzed. (3) In situ end-terminal transferase (TUNEL) and immunohistochemical method, the apoptosis of lung cells was detected. and analyze uPA system 3. In vitro cell experiment (1), the immortalized human type 鈪，
本文编号：2487369
[Abstract]:Study Background Chronic obstructive pulmonary disease (COPD) is a common and multiple disease that is harmful to human health. It has a high rate of disability, high mortality, and heavy economic and social burden, which seriously reduces the quality of life of patients and their families. The quantity is a public health question that every society and government must face and solve At present, people's understanding of the nature of COPD is not enough, especially for the causes and mechanisms of the main pathological changes, and the understanding of the mechanism is rather superficial, leading to the diagnosis and prevention of COPD. In 1957, a scholar suggested that the pathological changes of COPD started with the small airway, and the subsequent study constantly confirmed the weight of the small airway disease in the pathogenesis of COPD. To be in position. The most thought-provoking of the pathological change in COPD is the coexistence of such hyperplasia and degradation of the tissue destruction of small-wall collagen deposition and emphysema The study and understanding of this distinct pathological outcome is of great significance for elucidating the pathogenesis and establishing a scientific and efficient diagnosis and treatment method. It is of great significance that the expression pattern of the extracellular matrix in the synthesis/ degradation of the extracellular matrix is one of the causes of different pathological changes in the airway region and the alveolar region of the COPD, and at the same time, the role of the apoptosis in the formation of the emphysema is also increasing. Concern is expressed that the balance of extracellular matrix synthesis/ degradation and cell apoptosis/ proliferation may be co-involved in COPD For COPD patients, some of the systems in their body or lung tissue have an abnormal change in the long-term effects of environmental or pathogenic factors, leading to COPD The pathological changes of urokinase-type plasminogen activator (uPA) in the inflammation and tissue remodeling of the patients with COPD have been confirmed in our early studies. in addition to that fibrinolytic function, the urokinase-type plasminogen activator system has various functions of non-fibrinolysis, such as regulation of cell adhesion, differentiation, movement, apoptosis/ proliferation, and the like, and is involved in various diseases The purpose of this study is to explore the role of urokinase-type plasminogen activator system in the formation of small airway collagen in COPD and the formation of emphysema, and to explain the pathological changes and mechanism of the COPD from a new point of view. supply Objective To study the target of uPA system in the pathological changes of COPD. 1. To observe the molecular expression pattern of the uPA system in the lung tissue of patients with COPD and to analyze the molecular expression pattern of the uPA system in the lung tissue of patients with COPD. the relationship between the pathological changes of COPD;2. Clear the component and COP of uPA system 3. The relationship between the apoptosis of lung tissue cells in patients with D.3. The urokinase-type plasminogen activator inhibitor (PAI)-1, one of the main components of the uPA system, was expressed in COPD. alveoli epithelial cell apoptosis Methods 1. The cases were selected to select 36 patients with pulmonary lobectomy for benign lesions, and the lung function and CT were performed in accordance with the Global Initiative for Chronic Obstructive Pulmonary Disease (GOL). D) The diagnosis standard of the patients was divided into 16 cases of COPD group and 20 cases of normal control group (n = 20). The smoking control group (10 cases) Non-smoking control group (n = 10).2. The detection of tissue specimen in the city (1) the detection of the tissue specimen of the city (1) the detection of uPA, urokinase-type plasminogen activator receptor (uPAR), and urinary shock in the lung tissue. Expression of the enzyme-type plasminogen activator (PAI-1) and PAI-2. (2) The relationship between the expression of uPA system components in the lung tissue and the collagen deposition of the small airway wall was analyzed by the staining of the tissue plasminogen activator (PAI-1) and the tissue plasminogen activator (PAI-1). The relationship between the expression of uPA system and the index of emphysema in lung tissue was analyzed. (3) In situ end-terminal transferase (TUNEL) and immunohistochemical method, the apoptosis of lung cells was detected. and analyze uPA system 3. In vitro cell experiment (1), the immortalized human type 鈪，
本文编号：2487369
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