尿激酶型纤溶酶原激活物及其受体在COPD小气道上皮EMT中作用的研究

发布时间：2018-04-21 02:39

  本文选题：慢性阻塞性肺疾病 + 尿激酶型纤溶酶原激活物　； 参考：《山东大学》2014年博士论文

【摘要】：研究背景 慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)是一种常见病和多发病。在世界范围内患病率和死亡率都很高,经济社会负担重,已成为一个重要的公共卫生问题。据世界银行／世界卫生组织公布,至2020年COPD将位居世界疾病经济负担排名的第5位。COPD严峻的发病形势及其对人类健康所造成的重大威胁缘于人们对COPD的认识不足。加强并深入COPD的研究,特别是对其病理改变相关机制进行探讨,将有助于寻找COPD防治的新靶点,制定更切实有效的防治策略。 早在半个多世纪以前,Spain DM[1]等就提出：COPD气流受限和气道狭窄的部位起始于小气道,其本质是小气道壁炎症和纤维化。但由于小气道(内径2mm)特殊的病理解剖和病理生理的特点,以及研究方法和技术的受限,在此后的几十年里,对于COPD小气道发病机理的研究进展缓慢。近年来,随着细胞和分子生物学、分子病理技术、实验动物学的发展以及新的技术和实验方法的出现,COPD小气道的研究进入了一个新阶段。 小气道壁增厚是COPD的重要病理改变,并且与COPD呼气气流受限密切相关[2]。小气道周围肌成纤维细胞聚集是导致小气道壁增厚的原因之一。小气道周围肌成纤维细胞主要来源于：①小气道周围成纤维细胞；②小气道平滑肌细胞；③循环中的成纤维干细胞；④上皮间质转化(epithelial-mesenchymal transition, EMT)。 EMT是指极化的上皮细胞经历多种生物化学的改变最终表现出间质细胞特性的过程。多项研究证实,EMT与多种器官纤维化的发生、发展有着密切的关系。近年来,EMT在COPD疾病发生发展中的作用日益为研究者所重视[3]。新近研究表明,EMT在COPD患者小气道上皮明显存在,且与COPD的主要危险因素吸烟密切相关,经历了EMT的小气道上皮细胞能穿透基底膜迁移到黏膜下层转化成成纤维细胞/肌成纤维细胞。这些结果显示小气道上皮细胞EMT的发生是COPD小气道壁增厚和纤维化的重要因素。然而迄今为止,COPD小气道上皮细胞EMT的研究仅限于现象的描述,其发生机制鲜有研究报道。 我们前期研究证实了尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator, uPA)及其受体(uPA receptor, uPAR)在COPD的组织重塑中有重要作用。那么,uPA及uPAR对于COPD小气道上皮细胞EMT的发生是否具有作用?如有作用,其机制是什么?本研究试图从人肺组织标本和体外细胞实验两个层面来探讨uPA及uPAR在COPD小气道上皮EMT发生中的作用,从一个新的角度阐述COPD小气道重塑的病理机制,为COPD的有效防治提供新的靶点。 研究目的 探讨uPA和uPAR在COPD小气道上皮EMT中的作用及其相关分子机制 方法 1.通过COPD患者肺组织标本观察小气道上皮EMT状况及其与uPA/uPAR之间的关系 (1)病例选择：选择因肺部病变行肺叶切除术的患者78例,术前行肺功能检查,按照慢性阻塞性肺疾病全球倡议(GOLD)2011修订版的诊断标准及吸烟史,将患者分为COPD组,肺功能正常对照组(其中分为对照吸烟组和对照不吸烟组)。 (2)通过免疫组织化学方法检测肺组织标本小气道上皮EMT上皮细胞分子标志物E-cadherin和间质细胞分子标志物vimentin, uPA和uPAR的表达。 (3)结合患者肺功能资料,分析肺组织小气道上皮间质细胞标志物vimentin, uPA和uPAR的表达与FEV1%的相关性；分析肺组织小气道上皮uPA和uPAR的表达与间质细胞标志物vimentin表达的相关性。 2.体外培养人小气道上皮细胞HSAEpiCs研究香烟提取物(cigarette smoke extract, CSE)对小气道上皮EMT的影响,阐明uPA及uPAR在小气道上皮细胞EMT中的作用,探讨其分子机制。 (1)CSE对HSAEpiCs细胞EMT的影响：①通过倒置相差显微镜观察细胞形态变化；②通过qRT-PCR、Western Blot方法检测上皮细胞标志物E-cadherin和a-catenin,间质细胞标志物N-cadherin和a-SMA的表达；③通过Transwell方法检测CSE对HSAEpiCs细胞迁移的影响。 (2)uPAR在CSE诱导HSAEpiCs细胞EMT中的作用及分子机制研究： ①CSE对HSAEpiCs细胞uPAR表达的影响：通过qRT-PCR. Western Blot方法检测uPAR的表达变化。 ②沉默uPAR基因表达对CSE诱导HSAEpiCs细胞EMT的影响：通过RNA干扰技术沉默uPAR表达,采用qRT-PCR和Western Blot方法分别验证uPAR的表达；通过倒置相差显微镜观察沉默uPAR基因表达后对细胞形态的影响；通过Western blot方法检测干扰uPAR基因表达后EMT相关标志物的变化。 (3) PI3K/Akt信号通路对uPAR诱导的HSAEpiCs细胞EMT的影响：通过Western Blot方法检测CSE对p-Akt表达的影响；通过Western Blot方法检测沉默uPAR表达对PI3K/Akt信号通路的影响；通过Western Blot方法检测LY294002(PI3K抑制剂)对EMT相关标志物表达的影响。 (3)uPA在CSE诱导HSAEpiCs细胞EMT中的作用及分子机制研究： ①CSE对HSAEpiCs细胞uPA表达的影响：通过qRT-PCR、Western Blot方法检测uPA的表达变化。 ②抑制uPA表达对CSE诱导HSAEpiCs细胞EMT的影响：氨氯吡咪(uPA抑制剂)抑制uPA表达,通过倒置相差显微镜观察抑制uPA表达对细胞形态的影响；通过Transwell方法检测抑制uPA表达对CSE诱导HSAEpiCs细胞迁移能力的影响；通过Western Blot方法检测抑制uPA表达对CSE诱导HSAEpiCs细胞EMT相关标志物的变化。 ③uPA过表达对HSAEpiCs细胞EMT的影响：转染高表达uPA质粒,通过Western Blot方法验证uPA表达；通过倒置相差显微镜观察uPA过表达对细胞形态的影响；通过Transwell方法检测uPA过表达对HSAEpiCs细胞迁移能力的影响；通过Western Blot方法检测uPA过表达对HSAEpiCs细胞EMT相关标志物的变化。 ④沉默uPAR基因表达对uPA过表达诱导HSAEpiCs细胞EMT的影响：通过Western Blot方法验证沉默uPAR基因表达对uPA过表达诱导HSAEpiCs细胞EMT相关标志物的变化。 结果 1. COPD患者小气道上皮EMT标记物、uPA. uPAR表达及其它们之间的相关性分析 (1)小气道上皮EMT标记物的表达：COPD患者小气道上皮细胞E-cadherin表达降低,相应部位的间质细胞标志物vimentin表达增加；COPD组、对照吸烟组患者小气道上皮vimentin阳性染色细胞数较对照不吸烟组明显增多；COPD吸烟组、COPD不吸烟组患者小气道上皮vimentin阳性染色细胞数较对照吸烟组比较表达增多；COPD吸烟组小气道上皮vimentin阳性染色细胞数同COPD不吸烟组比较无显著差异。 (2)小气道上皮uPA和uPAR的表达：uPAR蛋白在COPD组、对照吸烟组患者小气道上皮细胞表达显著高于对照不吸烟组；COPD组小气道上皮细胞uPAR蛋白与对照吸烟组比较表达明显升高；uPAR在COPD吸烟组与COPD不吸烟组小气道上皮细胞表达无显著差异；uPA蛋白在COPD组、对照吸烟组患者小气道上皮细胞表达显著高于对照不吸烟组；COPD组小气道上皮细胞uPA蛋白显著高于对照吸烟组。 (3)小气道上皮EMT标记物、uPA和uPAR的表达与肺功能之间的相关性分析：小气道上皮vimentin阳性染色细胞数、uPAR和uPA表达与FEV1/预计值%呈显著负相关；小气道上皮uPAR、uPA蛋白表达与间质细胞标志物vimentin阳性染色细胞数呈显著正相关。 2.uPA及uPAR在CSE诱导小气道上皮细胞EMT中的作用及其分子机制 (1)CSE对HSAEpiCs细胞EMT的作用：CSE干预后,HSAEpiCs细胞由鹅卵石状上皮形态变成梭形、纺锤形；上皮细胞标志物E-cadherin、α-catenin表达下调,而间质细胞标志物N-cadherin和a-SMA表达上调；HSAEpiCs细胞迁移能力明显增强。 (2) uPAR在CSE诱导HSAEpiCs细胞EMT中的作用及分子机制研究： ①CSE对HSAEpiCs细胞uPAR表达的影响：CSE干预后显著增加uPAR的表达； ②沉默uPAR基因表达对CSE诱导HSAEpiCs细胞EMT的影响：沉默uPAR基因表达的HSAEpiCs细胞在CSE干预后细胞呈鹅卵石状的上皮形态；沉默uPAR基因表达能抑制CSE诱导的上皮细胞标志物E-cadherin、α-catenin蛋白表达下调和间质细胞标志物N-cadherin和a-SMA蛋白表达上调。 ③PI3K/Akt信号通路对uPAR诱导的HSAEpiCs细胞EMT的影响：CSE干预后,p-Akt表达升高；uPAR基因沉默后,p-Akt及下游信号分子表达显著降低,尤其在CSE干预组,给予PI3K抑制剂LY294002后,能降低CSE诱导的PI3K/Akt信号通路活化,并抑制EMT的发生。 (3)uPA在CSE诱导HSAEpiCs细胞EMT中的作用及分子机制研究： ①CSE对HSAEpiCs细胞uPA表达的影响：CSE干预后显著增加uPA的表达。 ②抑制uPA表达对CSE诱导HSAEpiCs细胞EMT的影响：氨氯吡咪抑制uPA表达后,在CSE干预后细胞保持鹅卵石状的上皮形态；氨氯吡咪抑制CSE诱导HSAEpiCs细胞迁移能力增强；氨氯吡咪抑制CSE诱导HSAEpiCs细胞EMT。 ③uPA过表达对HSAEpiCs细胞EMT的影响：uPA过表达促进细胞由上皮细胞形态向间质细胞形态转变；uPA过表达促进HSAEpiCs细胞迁移能力增强；uPA过表达促进HSAEpiCs细胞EMT。 ④沉默uPAR基因表达对uPA过表达诱导HSAEpiCs细胞EMT的影响：沉默uPAR基因表达能部分逆转uPA过表达诱导的HSAEpiCs细胞EMT。 结论 1.COPD患者小气道上皮存在明显EMT现象,并且分别与高表达的uPA和uPAR密切相关。 2. uPAR对小气道上皮细胞EMT具有调控作用,其可通过PI3K/Akt信号通路调控EMT发生。 3.uPA对小气道上皮细胞EMT同样具有调控作用,部分依赖于uPAR的表达。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R563.9

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