【作者】 王淑娟；

【导师】 董亮；

【作者基本信息】 山东大学 ， 内科学， 2008， 硕士

【摘要】 目的:支气管哮喘（简称哮喘）是由多种细胞（如嗜酸性粒细胞、T淋巴细胞、气道上皮细胞等）和细胞组份参与的气道炎症性疾患。其主要病理特征是急慢性气道炎症,平滑肌功能紊乱和气道重塑,是危害人类健康的常见病和多发病。早期研究认为哮喘是完全可逆性疾病,然而目前研究显示哮喘的自然过程并非完全可逆。其根本原因可能是部分哮喘患者发生了早期的气道重塑。气道重塑的定义主要是以组织学描述为主,其中转变表型的肌成纤维细胞和胶原沉积是早期气道重塑的重要特点。此类转变表型的肌成纤维细胞能够表达α-平滑肌动蛋白（α-SMA）,α-SMA是肌纤维细胞亚类之一,具有收缩潜能和较强的胶原合成能力,可导致管腔收缩。同时转变表型的肌成纤维细胞也是肺内胶原合成的主要细胞,它的聚集可促进胶原等细胞外基质的过度沉积,尤其是Ⅰ型胶原,促使管壁增厚。所以α-SMA和Ⅰ型胶原被认为是判断气道重塑的重要指标。现已知哮喘早期气道重塑是多基因参与的结果,大量细胞因子活化,促进炎症基因表达,引起气道高反应性和气道结构改变,致使哮喘早期气道重塑形成。此过程中炎症基因“发现于炎症区分子1”（FIZZ1）的作用和功能备受关注。FIZZ1或RELM-α是2000年首次发现的独立新基因,命名为“发现于感染区”或者“类抵抗素分子”。其在肺和心脏等器官中特异性表达,并且在过敏性疾病中显著高表达。同时FIZZ1也在小鼠慢性低氧肺模型中显著高表达,因此FIZZ1也被称为低氧诱导的有丝分裂因子（HIMF）。研究显示FIZZ1在肺间质纤维化动物模型的气道上皮细胞和肺泡Ⅱ型上皮细胞（AECⅡ）中特异性高表达,并且诱导肌成纤维细胞分化及细胞外基质的沉积。现已知哮喘患者早期气道重塑也就是气道纤维化的过程,因此,上述研究为哮喘气道重塑过程提供了重要的线索。因此我们提出如下假说:FIZZ1可能是早期气道重塑中重要的炎症基因,诱导α-SMA和Ⅰ型胶原等气道重塑的重要指标高表达。本研究拟通过探讨支气管哮喘大鼠肺组织Ⅱ型肺泡上皮细胞FIZZ1的表达水平,并观察肺支气管内早期气道重塑的表现。同时运用体外转染FIZZ1基因的方法,进一步证实FIZZ1对α-SMA和Ⅰ型胶原等早期气道重塑主要指标的直接调节作用,描述炎症基因FIZZ1与早期气道重塑的关系。本研究通过揭示炎症基因FIZZ1在哮喘发病过程中的作用,进一步阐述哮喘的发病机制。同时,也为寻求阻断早期气道重塑和不可逆气流受限的方法,提供了理论依据。因此研究并应用FIZZ1阻断剂,将是一种很有前景的针对性治疗策略。方法:健康WT大鼠70只,将大鼠随机分为生理盐水组（saline group）,OVA哮喘组（OVA group）等各35只。复制大鼠哮喘模型;检测大鼠的肺功能指标:气道阻力（Re）和肺动态顺应性（Cldyn）;分离两组肺组织中Ⅱ型肺泡上皮细胞（AECⅡ）;采用逆转录-聚合酶链反应（RT-PCR）法检测细胞内FIZZ1mRNA的表达强度;免疫组化法测定肺支气管中α-SMA表达水平;对肺成纤维细胞体外转染pEGFP-FIZZ1融合质粒和空载体质粒,并且加入TGF-β1抗体,排除其对肌成纤维细胞的诱导作用;采用酶联免疫吸附法（ELISA）测定成纤维细胞提取物中的Ⅰ型胶原表达水平,同时采用免疫印迹法（western blotting）测定成纤维细胞提取物中α-SMA表达强度。统计学分析:所有数据均以（均数±标准差）表示,采用SPSS10.0软件处理。样本间均数的比较采用t检验。P＜0.05表示有统计学差异,P＜0.01表示有显著统计学差异。x线片和电泳图采用AlphaImager 2200图象分析软件,免疫组化图片采用Image-prol-plus（美国马里兰公司生产）软件分析。结果:1.大鼠造模成功的临床表现以大鼠出现烦躁不安、活动频繁、呼吸急促、缩胸收腹,上肢抬起,点头呼吸,以及大小便失禁等为诱喘成功的标志。2.大鼠肺气道阻力（Re）和肺动态顺应性（Cldyn）的测定吸入浓度为0.025 mg/kg的ACH后,两组大鼠的气道阻力和肺动态顺应性的实测值/基础值基本无差别（P=0.379,P=0.843）;ACH浓度的提高到（0.05-0.2mg/kg）,OVA哮喘组气道阻力的实测值/基础值增高比例明显高于生理盐水组（P＜0.05）,同时OVA哮喘组肺动态顺应性的实测值/基础值显著低于生理盐水组（P＜0.05）。3.HE染色观察肺部炎症表现病理证实造模成功:大鼠OVA哮喘组中可见肺支气管上皮细胞脱落坏死,气管腔内大量炎性细胞浸润,包括嗜酸性粒细胞,T淋巴细胞等,以及管壁增厚,管腔狭窄等炎症表现。4.免疫组化检测肺组织内支气管α—SMA的表达水平大鼠OVA哮喘组中可见肺支气管壁上存在大量被染成褐色α—SMA阳性免疫反应,OVA哮喘组发生了气道重塑;生理盐水组中可见α—SMA免疫反应信号呈阴性反应,仅可见到少数肺内血管壁平滑肌呈弱阳性,未发生明显的气道重塑(P=1.49×10^-5)。5.FIZZ1 mRNA在Ⅱ型肺泡上皮细胞的表达水平肺泡Ⅱ型上皮细胞呈立方形,相互连接镶嵌成片,呈岛形分布。OVA哮喘组肺组织中FIZZ1 mRNA在AECⅡ中的表达强度较生理盐水组显著增强,具有统计学意义(P=2.71×10^-8)。6.FIZZ1 mRNA的表达强度与α-SMA的相关性肺泡Ⅱ型上皮内FIZZ1 mRNA的表达强度与肺组织中α-SMA呈线性正相关(r=0.95,P=3.24×10^-6)。7.肺内成纤维细胞中Ⅰ型胶原的表达水平转染pEGFP-FIZZ1质粒的转染组中纤维细胞提取物中的Ⅰ型胶原含量显著高于转染空载体质粒的对照组,两组差异具有统计学意义(P=3.64×10^-4)。8.肺内成纤维细胞中α-SMA的蛋白表达水平转染组成纤维细胞提取物中α-SMA的蛋白表达水平显著增强,而对照组无明显表达,两组差异具有统计学意义（P=0.0065）。结论:1.FIZZ1在哮喘动物模型Ⅱ型肺泡上皮细胞内特异性高表达,同时诱导哮喘模型支气管壁α-SMA蛋白的高表达。2.FIZZ1可诱导肌成纤维细胞分化,促使哮喘早期气道重塑特异性指标α-SMA和Ⅰ型胶原表达增强。证实FIZZ1是能够独立引起哮喘早期气道重塑的重要炎症因子之一。更多还原

【Abstract】 Objectives:Asthma is a chronic airway disease with inflammation characterized by airway hyperresponsiveness（AHR）and pathological changes features with the dysfunction of the smooth muscle and the deposition of extracellular matrix.Several lines of evidence suggested that not only severe asthma but also mild and moderate asthma were sometimes difficult to control by routine treatments,due to the early stage of airway remodeling in lung.The early stage airway remodeling in asthma thus might predispose persons with asthma to asthma exacerbations and even death from airway obstruction caused by smooth muscle contraction,airway edema,and mucus plugging.There were evidences from studies demonstrating that the occurrences of early stage airway remodeling in asthma were associated with the pulmonary myofibroblast differentiation,which was known to be a critical modulator ofα-smooth muscle actin（α-SMA）.Furthermore,bronchial biopsies of asthmatic patients showed thickening of the subepithelial layer due to the deposition of fibrillar collagen,such as elastin,collagenⅠ,and collagenⅢ.Thus,α-SMA and typeⅠcollage were regards as the main parameters in asthma airway remodeling.So the early stage airway remodeling was a hallmark of the clinical symptom of asthma, which could cause irreversible airflow limitation.Furthermore,Liu et al has provided some clues as to the potential role of certain genes in pathogenesis of airway remodeling.FIZZ1 was highly induced in lung fibrosis,and high FIZZ1-expressing AECⅡsignificantly stimulated both collagen typeⅠandα-SMA expression in fibroblasts.Therefore,both of these endpoints are reflective of fibroblast activation and differentiation to myofibroblasts,which were thought to share the resemblance pathogenesis of airway remodeling.We used pEGFP-FIZZ1 to transfect lung fibroblasts independent of TGF-β1 in order to provide more indirect evidences to elucidate the function of FIZZ1 in airway remodeling process.The myofibroblasts were major producers of collagenous matrix molecules which correlate with subepithelial collagen deposition andα-SMA.Cumulated evidences showed that the appearance ofα-SMA was correlated with the extent of airflow obstruction and likely contributed to airway wall thickening,at the same time collagen deposition caused the structural alterations in the airway,finally leading to airway collapse and airflow limitation.These findings provided valuable information in further understanding of FIZZ1 signal transduction pathways,which may ultimately lead to recognize more clearly mechanism of airway remodeling in asthma.Taken together, these results indicated a novel and critical function of FIZZ1 on the pathogenesis of airway remodeling.Methods:70 rats were divided into 2 groups:saline group 35 and OVA group 35. Pulmonary function tests were performed to evaluate the airway responsiveness;Isolation and culture the AECⅡ;The concentration of FIZZ1mRNA in AECⅡwere measured by the RT-PCR;Theα-SMA expression level were measured by immunohistochemistry;The lung fibroblast（LF）were transfected by pEGFP-FIZZ1 independent of TGF-β1;The expression of typeⅠcollagen were determined by ELISA.Theα-SMA in the LF expression was measured by western blotting.Results:1.The Rat Asthma ModelStudies in the OVA-induced model had demonstrated that a subset of overaction as a major features of asthma successful model.When compared with rats in saline group,the rats from OVA group showed dysphoria,more activity,upper limb raise, urinary and fecal incontinence and so on.2.Pulmonary function testsWhen stimulation of ACH with concentrations（0.025 mg/kg）,the mean Re and Cldyn of two groups have no difference（P=0.379,P=0.843）.After stimulation of ACH with accelerated concentrations（0.05-0.2mg/kg mg/kg）,the mean expiratory resistance（Re）of OVA group was higher than that of saline group（P＜0.05）.After stimulation of ACH with accelerated concentrations（0.05-0.2mg/kg mg/kg）,the mean Cldyn of OVA group was attenuate than that of saline group（P＜0.05）.3.Pato-manifestation by HE in Lung TissuesStudies in the OVA-induced model had demonstrated that a subset of inflammatory reaction as a major histopathological features of asthma.When compared with rats in saline group,the lung tissue from OVA treated rats showed wall thickening,inflammatory cells infiltration,subepithelial fibrosis,mucous metaplasia,and myocyte hyperplasia and hypertrophy.At the same time,we observed no obviously inflammatory reactions in saline group. 4.Immunohistochemical Staining ofα-SMA in Lung TissuesAs expected,positiveα-SMA staining was highly expressed in peribronchial lung sections isolated from the OVA group,and restricted to the muscular layer of the airway in the intrapulmonary bronchus.Neither the terminal bronchioles,nor the bronchial epithelia showed positive staining in saline group(P=1.49×10^-5).5.FIZZ1 mRNA Expression in AECⅡThe AECⅡisolated from lung had typical cube shape features under microscope. Previously studies demonstrated that FIZZ1 was specifically expressed in AECⅡof allergic pulmonary inflammation.When normalized toβ-actin,level of FIZZ1 mRNA was upregulated in AECⅡ,in contrast to very low level in control AECⅡ(P=2.71×10^-8).6.The relationship between the expression of FIZZ1 mRNA andα-SMAThe FIZZ1 mRNA expression has positive correlation with the peribronchial ofα-SMAin lung(r=0.95,P=3.24×10^-6).7.Expression of Collagen TypeⅠin Lung FibroblastBecause AECⅡwere shown to be a major source for FIZZ1 in OVA-induced asthma,their effect on certain fibroblast functions related to airway remodeling could be used to explore the potential role of this molecule in airway remodeling.In vitro,we tested the level of collagen typeⅠin lung fibroblast transfected with the pEGFP-FIZZ1 plasmid independent of TGF-β1 by ELISA.These results showed that FIZZ1 significantly activates rat lung fibroblasts with respect to typeⅠ collagen expression(P=3.64×10^-4).8.Expression ofα-SMA Protein in Lung FibroblastsThere were evidences showing that airway remodeling in asthma were associated with theα-SMA.In vitro,we tested the level ofα-SMA protein in lung fibroblasts transfected with the pEGFP-FIZZ1 plasmid independent of TGF-β1 by western blotting.α-SMA protein level in lung fibroblasts transfected with empty vector plasmid did not show any change.These results showed that FIZZ1 significantly activates rat lung fibroblasts with respect toα-SMA expression（P=0.0065）.Conclusion:1.FIZZ1 expressed higher in AECⅡand induced the emergance ofα-SMA in peribronchial lung sections.2.FIZZ1 induced myofibroblast differentiation to releaseα-SMA and typeⅠcollagen independent of TGF-β1,and FIZZ1 plays a critical function in the pathogenesis process of airway remodeling.更多还原