本文選題：慢性阻塞性肺疾病 + 尿激酶型纖溶酶原激活物　； 參考：《山東大學(xué)》2014年博士論文

【摘要】：研究背景 慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)是一種常見(jiàn)病和多發(fā)病。在世界范圍內(nèi)患病率和死亡率都很高,經(jīng)濟(jì)社會(huì)負(fù)擔(dān)重,已成為一個(gè)重要的公共衛(wèi)生問(wèn)題。據(jù)世界銀行／世界衛(wèi)生組織公布,至2020年COPD將位居世界疾病經(jīng)濟(jì)負(fù)擔(dān)排名的第5位。COPD嚴(yán)峻的發(fā)病形勢(shì)及其對(duì)人類(lèi)健康所造成的重大威脅緣于人們對(duì)COPD的認(rèn)識(shí)不足。加強(qiáng)并深入COPD的研究,特別是對(duì)其病理改變相關(guān)機(jī)制進(jìn)行探討,將有助于尋找COPD防治的新靶點(diǎn),制定更切實(shí)有效的防治策略。 早在半個(gè)多世紀(jì)以前,Spain DM[1]等就提出：COPD氣流受限和氣道狹窄的部位起始于小氣道,其本質(zhì)是小氣道壁炎癥和纖維化。但由于小氣道(內(nèi)徑2mm)特殊的病理解剖和病理生理的特點(diǎn),以及研究方法和技術(shù)的受限,在此后的幾十年里,對(duì)于COPD小氣道發(fā)病機(jī)理的研究進(jìn)展緩慢。近年來(lái),隨著細(xì)胞和分子生物學(xué)、分子病理技術(shù)、實(shí)驗(yàn)動(dòng)物學(xué)的發(fā)展以及新的技術(shù)和實(shí)驗(yàn)方法的出現(xiàn),COPD小氣道的研究進(jìn)入了一個(gè)新階段。 小氣道壁增厚是COPD的重要病理改變,并且與COPD呼氣氣流受限密切相關(guān)[2]。小氣道周?chē)〕衫w維細(xì)胞聚集是導(dǎo)致小氣道壁增厚的原因之一。小氣道周?chē)〕衫w維細(xì)胞主要來(lái)源于：①小氣道周?chē)衫w維細(xì)胞；②小氣道平滑肌細(xì)胞；③循環(huán)中的成纖維干細(xì)胞；④上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT)。 EMT是指極化的上皮細(xì)胞經(jīng)歷多種生物化學(xué)的改變最終表現(xiàn)出間質(zhì)細(xì)胞特性的過(guò)程。多項(xiàng)研究證實(shí),EMT與多種器官纖維化的發(fā)生、發(fā)展有著密切的關(guān)系。近年來(lái),EMT在COPD疾病發(fā)生發(fā)展中的作用日益為研究者所重視[3]。新近研究表明,EMT在COPD患者小氣道上皮明顯存在,且與COPD的主要危險(xiǎn)因素吸煙密切相關(guān),經(jīng)歷了EMT的小氣道上皮細(xì)胞能穿透基底膜遷移到黏膜下層轉(zhuǎn)化成成纖維細(xì)胞/肌成纖維細(xì)胞。這些結(jié)果顯示小氣道上皮細(xì)胞EMT的發(fā)生是COPD小氣道壁增厚和纖維化的重要因素。然而迄今為止,COPD小氣道上皮細(xì)胞EMT的研究?jī)H限于現(xiàn)象的描述,其發(fā)生機(jī)制鮮有研究報(bào)道。 我們前期研究證實(shí)了尿激酶型纖溶酶原激活物(urokinase-type plasminogen activator, uPA)及其受體(uPA receptor, uPAR)在COPD的組織重塑中有重要作用。那么,uPA及uPAR對(duì)于COPD小氣道上皮細(xì)胞EMT的發(fā)生是否具有作用?如有作用,其機(jī)制是什么?本研究試圖從人肺組織標(biāo)本和體外細(xì)胞實(shí)驗(yàn)兩個(gè)層面來(lái)探討uPA及uPAR在COPD小氣道上皮EMT發(fā)生中的作用,從一個(gè)新的角度闡述COPD小氣道重塑的病理機(jī)制,為COPD的有效防治提供新的靶點(diǎn)。 研究目的 探討uPA和uPAR在COPD小氣道上皮EMT中的作用及其相關(guān)分子機(jī)制 方法 1.通過(guò)COPD患者肺組織標(biāo)本觀察小氣道上皮EMT狀況及其與uPA/uPAR之間的關(guān)系 (1)病例選擇：選擇因肺部病變行肺葉切除術(shù)的患者78例,術(shù)前行肺功能檢查,按照慢性阻塞性肺疾病全球倡議(GOLD)2011修訂版的診斷標(biāo)準(zhǔn)及吸煙史,將患者分為COPD組,肺功能正常對(duì)照組(其中分為對(duì)照吸煙組和對(duì)照不吸煙組)。 (2)通過(guò)免疫組織化學(xué)方法檢測(cè)肺組織標(biāo)本小氣道上皮EMT上皮細(xì)胞分子標(biāo)志物E-cadherin和間質(zhì)細(xì)胞分子標(biāo)志物vimentin, uPA和uPAR的表達(dá)。 (3)結(jié)合患者肺功能資料,分析肺組織小氣道上皮間質(zhì)細(xì)胞標(biāo)志物vimentin, uPA和uPAR的表達(dá)與FEV1%的相關(guān)性；分析肺組織小氣道上皮uPA和uPAR的表達(dá)與間質(zhì)細(xì)胞標(biāo)志物vimentin表達(dá)的相關(guān)性。 2.體外培養(yǎng)人小氣道上皮細(xì)胞HSAEpiCs研究香煙提取物(cigarette smoke extract, CSE)對(duì)小氣道上皮EMT的影響,闡明uPA及uPAR在小氣道上皮細(xì)胞EMT中的作用,探討其分子機(jī)制。 (1)CSE對(duì)HSAEpiCs細(xì)胞EMT的影響：①通過(guò)倒置相差顯微鏡觀察細(xì)胞形態(tài)變化；②通過(guò)qRT-PCR、Western Blot方法檢測(cè)上皮細(xì)胞標(biāo)志物E-cadherin和a-catenin,間質(zhì)細(xì)胞標(biāo)志物N-cadherin和a-SMA的表達(dá)；③通過(guò)Transwell方法檢測(cè)CSE對(duì)HSAEpiCs細(xì)胞遷移的影響。 (2)uPAR在CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT中的作用及分子機(jī)制研究： ①CSE對(duì)HSAEpiCs細(xì)胞uPAR表達(dá)的影響：通過(guò)qRT-PCR. Western Blot方法檢測(cè)uPAR的表達(dá)變化。 ②沉默uPAR基因表達(dá)對(duì)CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT的影響：通過(guò)RNA干擾技術(shù)沉默uPAR表達(dá),采用qRT-PCR和Western Blot方法分別驗(yàn)證uPAR的表達(dá)；通過(guò)倒置相差顯微鏡觀察沉默uPAR基因表達(dá)后對(duì)細(xì)胞形態(tài)的影響；通過(guò)Western blot方法檢測(cè)干擾uPAR基因表達(dá)后EMT相關(guān)標(biāo)志物的變化。 (3) PI3K/Akt信號(hào)通路對(duì)uPAR誘導(dǎo)的HSAEpiCs細(xì)胞EMT的影響：通過(guò)Western Blot方法檢測(cè)CSE對(duì)p-Akt表達(dá)的影響；通過(guò)Western Blot方法檢測(cè)沉默uPAR表達(dá)對(duì)PI3K/Akt信號(hào)通路的影響；通過(guò)Western Blot方法檢測(cè)LY294002(PI3K抑制劑)對(duì)EMT相關(guān)標(biāo)志物表達(dá)的影響。 (3)uPA在CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT中的作用及分子機(jī)制研究： ①CSE對(duì)HSAEpiCs細(xì)胞uPA表達(dá)的影響：通過(guò)qRT-PCR、Western Blot方法檢測(cè)uPA的表達(dá)變化。 ②抑制uPA表達(dá)對(duì)CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT的影響：氨氯吡咪(uPA抑制劑)抑制uPA表達(dá),通過(guò)倒置相差顯微鏡觀察抑制uPA表達(dá)對(duì)細(xì)胞形態(tài)的影響；通過(guò)Transwell方法檢測(cè)抑制uPA表達(dá)對(duì)CSE誘導(dǎo)HSAEpiCs細(xì)胞遷移能力的影響；通過(guò)Western Blot方法檢測(cè)抑制uPA表達(dá)對(duì)CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT相關(guān)標(biāo)志物的變化。 ③uPA過(guò)表達(dá)對(duì)HSAEpiCs細(xì)胞EMT的影響：轉(zhuǎn)染高表達(dá)uPA質(zhì)粒,通過(guò)Western Blot方法驗(yàn)證uPA表達(dá)；通過(guò)倒置相差顯微鏡觀察uPA過(guò)表達(dá)對(duì)細(xì)胞形態(tài)的影響；通過(guò)Transwell方法檢測(cè)uPA過(guò)表達(dá)對(duì)HSAEpiCs細(xì)胞遷移能力的影響；通過(guò)Western Blot方法檢測(cè)uPA過(guò)表達(dá)對(duì)HSAEpiCs細(xì)胞EMT相關(guān)標(biāo)志物的變化。 ④沉默uPAR基因表達(dá)對(duì)uPA過(guò)表達(dá)誘導(dǎo)HSAEpiCs細(xì)胞EMT的影響：通過(guò)Western Blot方法驗(yàn)證沉默uPAR基因表達(dá)對(duì)uPA過(guò)表達(dá)誘導(dǎo)HSAEpiCs細(xì)胞EMT相關(guān)標(biāo)志物的變化。 結(jié)果 1. COPD患者小氣道上皮EMT標(biāo)記物、uPA. uPAR表達(dá)及其它們之間的相關(guān)性分析 (1)小氣道上皮EMT標(biāo)記物的表達(dá)：COPD患者小氣道上皮細(xì)胞E-cadherin表達(dá)降低,相應(yīng)部位的間質(zhì)細(xì)胞標(biāo)志物vimentin表達(dá)增加；COPD組、對(duì)照吸煙組患者小氣道上皮vimentin陽(yáng)性染色細(xì)胞數(shù)較對(duì)照不吸煙組明顯增多；COPD吸煙組、COPD不吸煙組患者小氣道上皮vimentin陽(yáng)性染色細(xì)胞數(shù)較對(duì)照吸煙組比較表達(dá)增多；COPD吸煙組小氣道上皮vimentin陽(yáng)性染色細(xì)胞數(shù)同COPD不吸煙組比較無(wú)顯著差異。 (2)小氣道上皮uPA和uPAR的表達(dá)：uPAR蛋白在COPD組、對(duì)照吸煙組患者小氣道上皮細(xì)胞表達(dá)顯著高于對(duì)照不吸煙組；COPD組小氣道上皮細(xì)胞uPAR蛋白與對(duì)照吸煙組比較表達(dá)明顯升高；uPAR在COPD吸煙組與COPD不吸煙組小氣道上皮細(xì)胞表達(dá)無(wú)顯著差異；uPA蛋白在COPD組、對(duì)照吸煙組患者小氣道上皮細(xì)胞表達(dá)顯著高于對(duì)照不吸煙組；COPD組小氣道上皮細(xì)胞uPA蛋白顯著高于對(duì)照吸煙組。 (3)小氣道上皮EMT標(biāo)記物、uPA和uPAR的表達(dá)與肺功能之間的相關(guān)性分析：小氣道上皮vimentin陽(yáng)性染色細(xì)胞數(shù)、uPAR和uPA表達(dá)與FEV1/預(yù)計(jì)值%呈顯著負(fù)相關(guān)；小氣道上皮uPAR、uPA蛋白表達(dá)與間質(zhì)細(xì)胞標(biāo)志物vimentin陽(yáng)性染色細(xì)胞數(shù)呈顯著正相關(guān)。 2.uPA及uPAR在CSE誘導(dǎo)小氣道上皮細(xì)胞EMT中的作用及其分子機(jī)制 (1)CSE對(duì)HSAEpiCs細(xì)胞EMT的作用：CSE干預(yù)后,HSAEpiCs細(xì)胞由鵝卵石狀上皮形態(tài)變成梭形、紡錘形；上皮細(xì)胞標(biāo)志物E-cadherin、α-catenin表達(dá)下調(diào),而間質(zhì)細(xì)胞標(biāo)志物N-cadherin和a-SMA表達(dá)上調(diào)；HSAEpiCs細(xì)胞遷移能力明顯增強(qiáng)。 (2) uPAR在CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT中的作用及分子機(jī)制研究： ①CSE對(duì)HSAEpiCs細(xì)胞uPAR表達(dá)的影響：CSE干預(yù)后顯著增加uPAR的表達(dá)； ②沉默uPAR基因表達(dá)對(duì)CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT的影響：沉默uPAR基因表達(dá)的HSAEpiCs細(xì)胞在CSE干預(yù)后細(xì)胞呈鵝卵石狀的上皮形態(tài)；沉默uPAR基因表達(dá)能抑制CSE誘導(dǎo)的上皮細(xì)胞標(biāo)志物E-cadherin、α-catenin蛋白表達(dá)下調(diào)和間質(zhì)細(xì)胞標(biāo)志物N-cadherin和a-SMA蛋白表達(dá)上調(diào)。 ③PI3K/Akt信號(hào)通路對(duì)uPAR誘導(dǎo)的HSAEpiCs細(xì)胞EMT的影響：CSE干預(yù)后,p-Akt表達(dá)升高；uPAR基因沉默后,p-Akt及下游信號(hào)分子表達(dá)顯著降低,尤其在CSE干預(yù)組,給予PI3K抑制劑LY294002后,能降低CSE誘導(dǎo)的PI3K/Akt信號(hào)通路活化,并抑制EMT的發(fā)生。 (3)uPA在CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT中的作用及分子機(jī)制研究： ①CSE對(duì)HSAEpiCs細(xì)胞uPA表達(dá)的影響：CSE干預(yù)后顯著增加uPA的表達(dá)。 ②抑制uPA表達(dá)對(duì)CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT的影響：氨氯吡咪抑制uPA表達(dá)后,在CSE干預(yù)后細(xì)胞保持鵝卵石狀的上皮形態(tài)；氨氯吡咪抑制CSE誘導(dǎo)HSAEpiCs細(xì)胞遷移能力增強(qiáng)；氨氯吡咪抑制CSE誘導(dǎo)HSAEpiCs細(xì)胞EMT。 ③uPA過(guò)表達(dá)對(duì)HSAEpiCs細(xì)胞EMT的影響：uPA過(guò)表達(dá)促進(jìn)細(xì)胞由上皮細(xì)胞形態(tài)向間質(zhì)細(xì)胞形態(tài)轉(zhuǎn)變；uPA過(guò)表達(dá)促進(jìn)HSAEpiCs細(xì)胞遷移能力增強(qiáng)；uPA過(guò)表達(dá)促進(jìn)HSAEpiCs細(xì)胞EMT。 ④沉默uPAR基因表達(dá)對(duì)uPA過(guò)表達(dá)誘導(dǎo)HSAEpiCs細(xì)胞EMT的影響：沉默uPAR基因表達(dá)能部分逆轉(zhuǎn)uPA過(guò)表達(dá)誘導(dǎo)的HSAEpiCs細(xì)胞EMT。 結(jié)論 1.COPD患者小氣道上皮存在明顯EMT現(xiàn)象,并且分別與高表達(dá)的uPA和uPAR密切相關(guān)。 2. uPAR對(duì)小氣道上皮細(xì)胞EMT具有調(diào)控作用,其可通過(guò)PI3K/Akt信號(hào)通路調(diào)控EMT發(fā)生。 3.uPA對(duì)小氣道上皮細(xì)胞EMT同樣具有調(diào)控作用,部分依賴于uPAR的表達(dá)。
[Abstract]:Research background
Chronic obstructive pulmonary disease (COPD) is a common and frequently occurring disease. The worldwide prevalence and mortality are high, the economic and social burden is heavy, and it has become an important public health problem. According to the world bank / WHO, by 2020, COPD will be the world's disease economy. The severe incidence of the fifth.COPD ranking and the major threat to human health is due to the lack of understanding of COPD. Strengthening and deepening the study of COPD, especially the mechanism of its pathological changes, will help to find new targets for the prevention and control of COPD and make more effective and effective prevention and control strategies.
As early as half a century ago, Spain DM[1], etc., suggested that the location of COPD airflow limitation and airway stenosis originate from the small airway, which is essentially small airway wall inflammation and fibrosis. But because of the special pathological and pathophysiological characteristics of the small airway (inner diameter 2mm), and the limitation of research methods and techniques, for the next few decades, The research on the pathogenesis of COPD small airway has been progressed slowly. In recent years, with the development of cell and molecular biology, molecular pathology, the development of experimental zoology and the emergence of new techniques and experimental methods, the study of small airway in COPD has entered a new stage.
The thickening of the wall of the airway is an important pathological change of COPD, and the aggregation of the myofibroblast around the small airway in [2]. is one of the reasons for the thickening of the wall of the small airway, which is closely related to the limitation of COPD expiratory air flow. Fibroblasts in the ring; epithelial-mesenchymal transition (EMT).
EMT refers to the process of polarized epithelial cells experiencing a variety of biochemical changes and the final expression of interstitial cell characteristics. A number of studies have confirmed that EMT has a close relationship with the development of multiple organ fibrosis. In recent years, the role of EMT in the development of COPD disease has been increasingly valued by researchers in the recent study of [3]., and EMT in COP The small airway epithelium in D patients is obviously present and is closely related to the major risk factors of smoking in COPD. The small airway epithelial cells experienced through the EMT can penetrate the basement membrane to the submucosa and convert into fibroblast / myofibroblast. These results show that the occurrence of EMT in the small airway epithelial cells is the thickening of the COPD wall and the weight of fibrosis. However, up to now, the study of EMT in COPD small airway epithelial cells is limited to the description of the phenomenon, and its mechanism is rarely reported.
Our previous studies have confirmed that the urokinase-type plasminogen activator (uPA) and its receptor (uPA receptor, uPAR) play an important role in the tissue remodeling of COPD. Then, whether uPA and uPAR have a role in the occurrence of EMT by COPD small airway epithelial cells? What is the mechanism? The role of uPA and uPAR in the occurrence of EMT in COPD small airway epithelium is discussed from two levels of human lung tissue specimens and in vitro cell experiments. The pathological mechanism of COPD small airway remodeling is described from a new perspective, which provides a new target for the effective prevention and control of COPD.
research objective
To investigate the role of uPA and uPAR in EMT of COPD small airway epithelium and its related molecular mechanisms.
Method
1. observe the EMT status of small airway epithelium and its relationship with uPA/uPAR through lung tissue specimens from COPD patients.
(1) case selection: 78 cases of pulmonary lobectomy were selected. Pulmonary function examination was performed before operation. According to the diagnostic criteria and smoking history of the 2011 revised version of GOLD, the patients were divided into COPD group and normal control group (which were divided into the control smoking group and the control non smoking group).
(2) the expression of the molecular marker of EMT epithelial cells in the small airway epithelium, E-cadherin, and the molecular markers of interstitial cells, vimentin, uPA and uPAR, were detected by immunohistochemistry.
(3) to analyze the correlation between the expression of interstitial cell markers vimentin, uPA and uPAR and the correlation between the expression of uPA and uPAR and the expression of uPA and uPAR in the small airway epithelium and the expression of interstitial cell marker vimentin in the small airway epithelial cells of the lung tissue.
2. the effect of cigarette smoke extract (CSE) on the EMT of small airway epithelium was studied by HSAEpiCs in vitro culture of human small airway epithelial cells, and the role of uPA and uPAR in the EMT of small airway epithelial cells was elucidated and its molecular mechanism was discussed.
(1) the effect of CSE on the EMT of HSAEpiCs cells: (1) the morphological changes of cells were observed by inverted phase contrast microscope; (2) the expression of E-cadherin and a-catenin of epithelial cell markers was detected by qRT-PCR, Western Blot method, and the expression of N-cadherin and a-SMA in interstitial cell markers; and the effect of CSE on the migration of cells was detected by Transwell method.
(2) the role and molecular mechanism of uPAR in CSE induced HSAEpiCs cell EMT.
The effect of CSE on the expression of uPAR in HSAEpiCs cells: the expression of uPAR was detected by qRT-PCR. Western Blot.
(2) the effect of silent uPAR gene expression on CSE induced EMT in HSAEpiCs cells: RNA interference technique was used to silence uPAR expression, qRT-PCR and Western Blot methods were used to verify the expression of uPAR, and the effect of the expression of the silent uPAR gene on the cell morphology was observed by the inverted phase microscope, and the interference gene was detected by the Western method. Changes in EMT related markers after expression.
(3) the effect of PI3K/Akt signaling pathway on the uPAR induced HSAEpiCs cell EMT: the effect of CSE on the expression of p-Akt through the Western Blot method, and the effect of the silent uPAR expression on the PI3K/Akt signal pathway through the Western Blot method; and the effect of the inhibitor on the expression of the related markers.
(3) the role and molecular mechanism of uPA in CSE induced HSAEpiCs cell EMT.
(1) the effect of CSE on the expression of uPA in HSAEpiCs cells: the expression of uPA was detected by qRT-PCR and Western Blot.
The effect of inhibition of uPA expression on CSE induced EMT in HSAEpiCs cells: the inhibition of uPA expression by amilamide (uPA inhibitor) and the effect of inhibition of uPA expression on the cell morphology by inverted phase contrast microscope, and the effect of the inhibition of uPA expression on the migration ability of CSE induced HSAEpiCs cells by Transwell method. Inhibition of uPA expression on EMT related markers in CSE induced HSAEpiCs cells.
(3) the effect of overexpression of uPA on the EMT of HSAEpiCs cells: transfection of high expression uPA plasmid, Western Blot method to verify uPA expression, the effect of uPA overexpression on cell morphology by inverted phase contrast microscope, and the effect of uPA overexpression on the migration energy of HSAEpiCs cells by Transwell method; Overexpression changes in EMT related markers in HSAEpiCs cells.
(4) the effect of silent uPAR gene expression on uPA overexpression induced EMT in HSAEpiCs cells: to verify the change of uPAR gene expression induced by uPA over expression induced HSAEpiCs cell EMT related markers by Western Blot method.
Result
1. the expression of EMT markers and uPA. uPAR in small airway epithelium of COPD patients and their correlation analysis
(1) expression of EMT markers in small airway epithelium: the expression of E-cadherin in small airway epithelial cells in COPD patients decreased and the expression of stromal cell marker vimentin in the corresponding parts increased; in COPD group, the number of vimentin positive cells in small airway epithelium in the control smoking group was significantly higher than that in the control non smoking group; COPD smoking group and COPD non-smoking group suffered from smoking group. The number of vimentin positive cells in the small airway epithelium in the small airway was more than that in the control smoking group, and there was no significant difference between the number of vimentin positive cells in the small airway epithelium in the COPD smoking group and the non smoking group in the COPD group.
(2) the expression of uPA and uPAR in the small airway epithelium: the expression of uPAR protein in group COPD, the expression of small airway epithelial cells in the control group was significantly higher than that in the control non smoking group; the expression of uPAR protein in the small airway epithelial cells in the COPD group was significantly higher than that in the control smoking group, and the expression of the small airway epithelial cells in the uPAR in the COPD smoking group and the non smoking group of COPD was not obvious. The expression of uPA protein in the COPD group and the control smoking group was significantly higher than that in the control group, and the uPA protein in the small airway epithelial cells in group COPD was significantly higher than that of the control smoking group.
(3) the correlation between the expression of EMT and the expression of uPA and uPAR with the pulmonary function: the number of vimentin positive staining cells in the small airway epithelium, the expression of uPAR and uPA was negatively correlated with the predictive value of FEV1/, and the expression of uPA protein in the small airway epithelium was positively correlated with the number of vimentin positive staining cells of the interstitial cell marker.
Role of 2.uPA and uPAR in CSE induced EMT in small airway epithelial cells and its molecular mechanism
(1) the effect of CSE on HSAEpiCs cell EMT: the prognosis of CSE stem, HSAEpiCs cells from cobblestone form into spindle shape, spindle shaped, E-cadherin, the expression of alpha -catenin down regulation, and the expression of N-cadherin and a-SMA in the interstitial cell markers, and the migration ability of HSAEpiCs cells obviously enhanced.
(2) the role and molecular mechanism of uPAR in CSE induced HSAEpiCs cell EMT.
(1) the effect of CSE on the expression of uPAR in HSAEpiCs cells: CSE significantly increased the expression of uPAR after intervention.
The effect of silent uPAR gene expression on CSE induced EMT in HSAEpiCs cells: HSAEpiCs cells expressed by silent uPAR gene were cobblestone shaped epithelial morphology after CSE intervention; silencing of uPAR gene expression could inhibit the CSE induced epithelial marker E-cadherin, alpha -catenin protein table downregulation and interstitial cell marker N-cadherin and The expression of a-SMA protein was up-regulated.
(3) the effect of PI3K/Akt signaling pathway on the EMT of HSAEpiCs cells induced by uPAR: the expression of p-Akt was increased after CSE, and the expression of p-Akt and downstream signal molecules decreased significantly after the uPAR gene was silenced, especially in the CSE intervention group. After the PI3K inhibitor LY294002, the activation of the CSE induced signaling pathway could be reduced and the occurrence of the uPAR was inhibited.
(3) the role and molecular mechanism of uPA in CSE induced HSAEpiCs cell EMT.
(1) the effect of CSE on the expression of uPA in HSAEpiCs cells: CSE significantly increased the expression of uPA after intervention.
(2) the effect of inhibition of uPA expression on CSE induced EMT in HSAEpiCs cells: after the inhibition of uPA expression by amilamide, the cells maintain cobblestone like epithelial morphology after CSE intervention; amlopiamide inhibits CSE induced migration ability of HSAEpiCs cells; amolimide inhibits CSE induced HSAEpiCs cell EMT..
(3) the effect of overexpression of uPA on the EMT of HSAEpiCs cells: uPA overexpression promotes the transformation of cells from epithelial cells to interstitial cells; uPA overexpression promotes the migration of HSAEpiCs cells; uPA overexpression promotes EMT. in HSAEpiCs cells.
(4) the effect of silent uPAR gene expression on uPA overexpression induced EMT in HSAEpiCs cells: silent uPAR gene expression can partly reverse the HSAEpiCs cell EMT. induced by uPA overexpression
conclusion
There is obvious EMT phenomenon in small airway epithelium of 1.COPD patients, and is closely related to the high expression of uPA and uPAR.
2. uPAR regulates EMT in small airway epithelial cells, which regulates EMT through PI3K/Akt signaling pathway.
3.uPA also regulates EMT in small airway epithelial cells, and partly depends on uPAR expression.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R563.9

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 許麗,汪濤,張珍祥,徐永健;粘著斑激酶活性對(duì)氣道上皮細(xì)胞粘附遷移的影響[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年03期

2 陳興無(wú),徐軍,鐘南山;氣道上皮損傷與上皮下成纖維細(xì)胞活化[J];國(guó)外醫(yī)學(xué).呼吸系統(tǒng)分冊(cè);2005年05期

3 馬燕;李娜萍;吳人亮;劉明閣;洪淵智;朱敏;田丹;;細(xì)胞質(zhì)連接蛋白-170在小鼠氣道上皮損傷修復(fù)過(guò)程中的動(dòng)態(tài)變化[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2007年01期

4 周曉婷;趙海金;蔡紹曦;;25羥維生素D_3對(duì)正常氣道上皮細(xì)胞通透性的影響及可能機(jī)制[J];南方醫(yī)科大學(xué)學(xué)報(bào);2011年07期

5 王山澤,葉曜芩,何慶,王曾禮;氣道上皮損傷及上皮衍化松弛因子與哮喘[J];國(guó)外醫(yī)學(xué).呼吸系統(tǒng)分冊(cè);1991年04期

6 王可;馮玉麟;文富強(qiáng);陳雪融;歐雪梅;徐丹;鄧治平;楊R，

本文編號(hào)：1780606