【摘要】：研究背景 慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)是一種危害人類(lèi)健康的常見(jiàn)病和多發(fā)病,其致殘率、死亡率高,經(jīng)濟(jì)和社會(huì)負(fù)擔(dān)重,嚴(yán)重降低病人和其家庭的生活質(zhì)量,是每一個(gè)社會(huì)和政府必須面對(duì)并要解決好的公共衛(wèi)生問(wèn)題。目前,人們對(duì)于COPD本質(zhì)的認(rèn)識(shí)還遠(yuǎn)遠(yuǎn)不夠,特別是對(duì)其主要的病理改變的原因和機(jī)制的了解還相當(dāng)膚淺,造成COPD的診斷和防治上的不盡人意。 早在1957年就有學(xué)者提出,COPD的病變起始于小氣道,隨后的研究不斷證實(shí)小氣道病變?cè)贑OPD發(fā)病中的重要地位。COPD病理改變最引人深思的是小氣道壁膠原沉積和肺氣腫的組織破壞的這種增生與降解共存的現(xiàn)象。研究和理解這種截然不同的病理結(jié)局,對(duì)于闡明發(fā)病機(jī)制,建立科學(xué)高效的診治方法具有重要意義。 晚近的研究提示,細(xì)胞外基質(zhì)合成/降解基因表達(dá)模式不同是造成COPD氣道區(qū)和肺泡區(qū)產(chǎn)生截然不同病理改變的原因之一；與此同時(shí),細(xì)胞過(guò)度凋亡在肺氣腫形成中的作用也日益引起人們的關(guān)注。細(xì)胞外基質(zhì)合成/降解和細(xì)胞凋亡/增殖的平衡失調(diào)可能共同參與了COPD的病理過(guò)程。對(duì)于COPD患者,其機(jī)體或肺組織中的某些系統(tǒng)在環(huán)境或致病因素的長(zhǎng)期作用下,發(fā)生了異常變化,而引發(fā)COPD的病理改變。我們的前期研究不斷證實(shí)了尿激酶型纖溶酶原激活物(urokinase-type plasminogen activator, uPA)系統(tǒng)成分在COPD的炎癥、組織重塑中有重要作用。尿激酶型纖溶酶原激活物系統(tǒng),除了纖溶功能外,更具有非纖溶的多種功能,諸如調(diào)節(jié)細(xì)胞粘附、分化、運(yùn)動(dòng)以及凋亡/增殖等,參與多種疾病病理調(diào)控過(guò)程。本研究試圖探究尿激酶型纖溶酶原激活物系統(tǒng)成分在COPD小氣道膠原沉積與肺氣腫形成中的作用,從一個(gè)新的角度闡釋COPD的病理改變及其機(jī)制,為COPD的治療提供新的靶點(diǎn)。 目的 探討uPA系統(tǒng)在COPD病理改變中的作用及其相關(guān)機(jī)制： 1.觀(guān)察COPD患者肺組織中uPA系統(tǒng)的分子表達(dá)模式,并分析其與COPD病理改變的關(guān)系； 2.明確uPA系統(tǒng)成分與COPD患者肺組織細(xì)胞凋亡的關(guān)系； 3.闡明uPA系統(tǒng)的主要成分之一尿激酶型纖溶酶原激活物抑制劑(PAI)-1在COPD肺泡上皮細(xì)胞凋亡中的作用及機(jī)制。 方法 1.病例選擇 選擇因良性病變行肺葉切除術(shù)的患者36例,術(shù)前行肺功能及CT檢查,按照慢性阻塞性肺疾病全球倡議(GOLD)的診斷標(biāo)準(zhǔn),將患者分為COPD組16例,肺功能正常對(duì)照組20例(其中吸煙對(duì)照組10例,不吸煙對(duì)照組10例)。 2.市組織標(biāo)本的檢測(cè) (1)免疫組織化學(xué)方法檢測(cè)肺組織標(biāo)本uPA、尿激酶型纖溶酶原激活物受體(uPAR)、尿激酶型纖溶酶原抑制劑(PAI-1)、PAI-2的表達(dá)。 (2)天狼猩紅染色檢測(cè)小氣道膠原沉積面積,分析肺組織中uPA系統(tǒng)成分表達(dá)與小氣道壁膠原沉積的相關(guān)性；結(jié)合患者術(shù)前胸部CT資料,分析肺組織中uPA系統(tǒng)成分表達(dá)與肺氣腫指數(shù)的相關(guān)性。 (3)原位末端轉(zhuǎn)移酶標(biāo)記技術(shù)(TUNEL)與免疫組織化學(xué)cleaved PARP染色,檢測(cè)肺組織細(xì)胞凋亡情況,并分析uPA系統(tǒng)成分在肺組織表達(dá)與肺組織細(xì)胞凋亡的相關(guān)性。 3.體外細(xì)胞實(shí)驗(yàn) (1)培養(yǎng)永生化人Ⅱ型肺泡上皮A549細(xì)胞系,應(yīng)用脂質(zhì)體2000將PAI-1小干擾RNA (siRNA)或無(wú)意義siRNA轉(zhuǎn)染到細(xì)胞中。 (2) Real-time PCR、western blot和ELISA方法檢測(cè)轉(zhuǎn)染前后PAI-1表達(dá),篩選出高效率抑制PAI-1表達(dá)的siRNA鏈。 (3)MTT方法檢測(cè)CSC對(duì)A549細(xì)胞存活率的影響,選擇合適濃度的CSC對(duì)轉(zhuǎn)染后細(xì)胞進(jìn)行刺激,模擬COPD細(xì)胞損傷過(guò)程。 (4) Annexin-Ⅴ和PI雙染流式細(xì)胞技術(shù)檢測(cè)細(xì)胞凋亡情況,探討PAI-1對(duì)肺泡上皮細(xì)胞凋亡的影響。 (5)化學(xué)發(fā)光法檢測(cè)轉(zhuǎn)染前后細(xì)胞Caspase3/7、Caspase8以及Caspase9的活性,探討PAI-1影響肺泡上皮細(xì)胞凋亡的通路。 (6)用Fas特異性中和抗體ZB4后重復(fù)轉(zhuǎn)染實(shí)驗(yàn)及凋亡檢測(cè),進(jìn)一步確定PAI-1影響肺泡上皮細(xì)胞凋亡的作用通路。 結(jié)果 1.肺組織標(biāo)本檢測(cè) (1)uPA系統(tǒng)各成分在小氣道區(qū)主要表達(dá)在小氣道上皮細(xì)胞,其中,COPD組uPA、uPAR與PAI-2在小氣道上皮細(xì)胞的表達(dá)均明顯高于對(duì)照組(P0.05)；而PAI-1在COPD組小氣道上皮細(xì)胞的表達(dá)低于對(duì)照組(P0.05)。在肺泡區(qū),uPA系統(tǒng)成分主要表達(dá)在肺泡上皮細(xì)胞和肺泡巨噬細(xì)胞,其中COPD組uPA、uPAR在肺泡上皮和肺泡巨噬細(xì)胞的表達(dá)均顯著高于對(duì)照組(P0.05),PAI-1在COPD組肺泡上皮細(xì)胞表達(dá)明顯低于對(duì)照組,而在COPD組與對(duì)照組肺泡巨噬細(xì)胞中的表達(dá)無(wú)顯著差異；PAI-2在COPD組與對(duì)照組肺泡上皮細(xì)胞與肺泡巨噬細(xì)胞表達(dá)均無(wú)顯著差異。 (2) COPD患者小氣道上皮uPA表達(dá)與氣道壁膠原沉積面積呈正相關(guān)(r=0.4187,P=0.0011),且肺泡上皮細(xì)胞uPA的表達(dá)與肺氣腫指數(shù)呈正相關(guān)(r=0.5188,p=0.0359)；COPD患者小氣道上皮細(xì)胞PAI-2的表達(dá)與氣道下膠原沉積面積呈正相關(guān)(r=0.5653,p=0.0225)。 (3) COPD組小氣道區(qū)凋亡指數(shù)(AI)較對(duì)照組無(wú)顯著差異,而肺泡上皮細(xì)胞AI較對(duì)照組顯著增高(P0.05)；COPD患者肺泡上皮PAI-1的表達(dá)與肺泡上皮AI呈負(fù)相關(guān)(r=-0.5122,P0.05)。 2.體外細(xì)胞實(shí)驗(yàn) (1) Real-time PCR、western bolt以及ELISA結(jié)果表明PAI-1siRNA可以顯著抑制PAI-1表達(dá),抑制率在90%以上。 (2)A549細(xì)胞存活率隨CSC濃度增加及刺激時(shí)間延長(zhǎng)而降低,具有濃度-時(shí)間-效應(yīng)關(guān)系。 (3) siRNA干擾A549細(xì)胞PAI-1表達(dá)后,細(xì)胞凋亡增多(P0.05)。加入5%CSC刺激后,相比單純干擾PAI-1,細(xì)胞凋亡也增多(P0.05)。 (4)干擾A549細(xì)胞PAI-1表達(dá)后,caspase3/7、caspase-8活性上調(diào)(P0.05),而caspase-9活性無(wú)明顯變化(P0.05)。 (5)給予Fas中和抗體(ZB4)后,干擾PAI-1細(xì)胞與無(wú)意義干擾細(xì)胞凋亡水平的差異消失,而給予非特異性IgG,干擾PAI-1細(xì)胞凋亡水平仍顯著高于無(wú)意義干擾細(xì)胞。 結(jié)論 1.uPA系統(tǒng)成分是參與COPD病理改變的重要物質(zhì)。 2.肺泡上皮細(xì)胞PAI-1低表達(dá)很可能是引起肺泡上皮細(xì)胞過(guò)度凋亡的原因之一。 3.PAI-1可以抑制肺泡上皮細(xì)胞的過(guò)度凋亡,其作用是通過(guò)Fas-FasL通路實(shí)現(xiàn)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，

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