【作者】 刘文；

【导师】 董亮；

【作者基本信息】 山东大学 ， 内科学（呼吸系病）（专业学位）， 2020， 博士

【摘要】 研究背景支气管哮喘(bronchial asthma)简称哮喘,是一种常见的慢性呼吸系统疾病,而气道高反应性、气道炎症及气道重塑是其三个重要的特征。目前针对气道炎症及气道高反应性的治疗方法可以有效地预防和缓解大多数的哮喘患者的临床症状,而一些经历持续不缓解的临床症状及肺功能进行性下降的哮喘被称为不可逆或难治性哮喘,对于这类患者目前尚缺少有效的治疗措施。气道重塑是哮喘的主要特征之一,这种重塑过程是严重哮喘的一个关键特征,被认为在难治性或持续性哮喘的发生发展中起到关键的作用。因此针对哮喘气道重塑机制的研究及针对气道重塑的各种组成部分的生物制剂的开发对于哮喘,尤其是难治性哮喘尤为必要。目前的动物模型、体外研究和一些临床研究已经提出了关于气道重塑中涉及的细胞和分子途径,这些途径的研究对于针对气道重塑的各类生物制剂的开发起到关键的作用,进而限制哮喘患者的气道重塑的发生,向哮喘的治愈迈了更近一步。结缔组织生长因子(Connective tissue growth factor,CTGF)是富含半胱氨酸的分泌肽。CTGF在OVA诱导的哮喘小鼠的肺组织中表达显著升高,同时与MMP-9、TIMP-1及平滑肌层的厚度呈正向相关,上述发现提示在哮喘气道重塑的发病机制中CTGF可能参与其中。进一步的研究发现在评估持续性气道阻塞程度时,血浆CTGF可能是稳定性哮喘的潜在生物标志物,CTGF可能与哮喘气道重塑有关。卵泡抑素样蛋白1(Follistatin-Like 1,Fstl1)属于卵泡抑素蛋白家族,它是一种细胞外基质的糖蛋白,属于分泌型蛋白,对于血管平滑肌细胞的增殖和迁移起到抑制作用,能够改善内皮细胞的功能,促进血管的再形成,在胚胎发育、器官形成、肿瘤、炎症反应中起到重要作用,同时对于心肌也能够起到保护作用。在哮喘气道重塑的发生发展中气道平滑肌细胞的异常增殖与迁移也起到重要的作用,它可以导致气道高反应性的发生,而研究已经证实在人气道平滑肌细胞的增殖与迁移过程中Fstl1起到重要的促进作用。在患有严重哮喘的患者的肺中,Fstl1被巨噬细胞高度表达,而敲除Fstl1能够抑制平滑肌细胞的迁移,提示在支气管哮喘的气道重塑的治疗研究中Fstl1可能代表了一个新的靶点。断开的相互作用蛋白 2 同源物 A(Disconnected(disco)-interacting protein 2 homolog A,DIP2A)是一种细胞质蛋白,DIP2A与FSTL1之间的直接物理相互作用已经被免疫共沉淀测定所证实,DIP2A作为FSTL1的受体介导FSTL1的下游信号通路。转化生长因子-β(Transforming growth factor beta,TGF-β)可以影响多种细胞的生长和分化,参与细胞的凋亡、免疫调节反应,并在上述过程中起到重要的作用。研究发现TGF-β的表达量在哮喘患者的气道组织中显著增加。TGF-β的许多作用由CTGF介导,CTGF是与TGF-β有关的关键的促纤维化因子之一。TGF-β是主要的组织纤维化的中心介质,而Fstl1则是在肺组织纤维化过程中重要的纤维前糖类蛋白,主要通过TGF-β信号通路促进纤维化。在肺成纤维细胞中TGF-β能诱导Fstl1生成,在小鼠肺纤维化过程中TGF-β上调Fstl1的表达。TGF-β的许多作用由CTGF介导,之前的研究已经明确FSTL1、CTGF均可以促进气道重塑,但是其具体作用机制尚不清楚。本研究以CTGF及FSTL1为切入点,旨在阐明其导致哮喘气道重塑的具体机制,为早期防治哮喘病人固定性气流受限提供临床治疗思路。研究目的探究CTGF是否通过影响FSTL1/DIP2A信号通路促进哮喘气道重塑的发生。研究方法1.明确CTGF及FSTL1在OVA诱导的小鼠过敏性气道疾病模型中起到重塑的作用。1.1采用6-8周雌性BALB/C小鼠,给予OVA诱导小鼠过敏性气道疾病模型,主要采用腹腔注射致敏和滴鼻吸入局部激发的方式。1.2造模成功后对各组小鼠进行气道反应性测定,给予不同浓度的乙酰甲胆碱雾化吸入测定并记录吸入后的气道阻力。1.3肺功能检测后处死并收集哮喘模型组及对照组小鼠的肺泡灌洗液、血液及肺组织标本。1.4对各组小鼠的肺泡灌洗液进行细胞的分类及计数。1.5测定各组小鼠外周血中IgE的浓度水平。1.6采用HE染色法分析气道重塑的特征性表现,包括气道上皮细胞的脱落、平滑肌束的增加以及基底膜增厚等。1.7采用Masson染色法分析小鼠气道中胶原纤维含量的表达。1.8应用免疫组织化学染色法测定各组CTGF、FSTL1的表达情况,同时测定起到重塑作用的相关标志物a平滑肌动蛋白(a-smooth muscle actin,a-SMA)、纤连蛋白及Ⅰ型胶原的表达。1.9应用Western blot方法测定CTGF、FSTL1及其受体DIP2A以及重塑相关蛋白的表达。1.10应用实时定量聚合酶链反应qRT-PCR方法检测CTGF、FSTL1及其受体DIP2A以及重塑相关标志物mRNA的表达。2.细胞水平观察CTGF对FSTL1/DIP2A信号通路的影响。2.1体外培养人肺成纤维细胞,给予FSTL1重组蛋白刺激后,检测DIP2A、a-SMA、纤连蛋白及Ⅰ型胶原的表达。2.2体外培养人肺成纤维细胞,给予CTGF重组蛋白刺激后,检测FSTL1、DIP2A、a-SMA、纤连蛋白及Ⅰ型胶原的表达。2.3体外培养人肺成纤维细胞,转染FSTL1 siRNA后给予CTGF重组蛋白处理,检测FSTL1、DIP2A、a-SMA、纤连蛋白及Ⅰ型胶原的表达。2.4体外培养人肺成纤维细胞,转染DIP2A siRNA后给予CTGF重组蛋白刺激后,检测FSTL1、DIP2A、a-SMA、纤连蛋白及Ⅰ型胶原的表达。2.5体外培养人肺成纤维细胞,转染DIP2A siRNA后给予FSTL1重组蛋白刺激后,检测CTGF、a-SMA、纤连蛋白及Ⅰ型胶原的表达。3.体内干预CTGF的表达对于哮喘小鼠FSTL1和气道重塑的影响。3.1采用6-8周雌性BALB/C小鼠,随机分为WT组、WT+OVA组、WT+OVA+Dex组、WT+OVA+CTGF组,WT+OVA组采用第一部分所描述的方法进行OVA哮喘小鼠模型制备,WT+OVA+Dex组在WT+OVA组模型制备的基础上腹腔注射地塞米松,WT+OVA+CTGF组在WT+OVA组模型制备的基础上滴鼻CTGF重组蛋白。3.2造模成功后对各组小鼠进行气道反应性的测定,测定并记录不同浓度下乙酰甲胆碱雾化吸入后的气道阻力。3.3肺功能检测结束后处死并收集各组小鼠的肺组织标本。3.4采用HE染色法观察各组小鼠的气道重塑的特征,包括上皮细胞的脱落、平滑肌束的增加及基底膜增厚等。3.5应用免疫组织化学法测定各组小鼠FSTL1的表达,同时测定起到重塑相关标志物a-SMA、纤连蛋白及Ⅰ型胶原的表达。3.6应用Western blot方法测定各组小鼠的FSTL1以及重塑相关标志物蛋白的表达。3.7应用实时定量聚合酶链反应qRT-PCR法检测各组小鼠的FSTL1以及重塑相关标志物mRNA的含量。结果1.哮喘小鼠模型的气道重塑表现明显,且CTGF、FSTL1含量增高。1.1 OVA诱导的哮喘小鼠模型组较对照组气道反应性增高。1.2 OVA诱导的哮喘小鼠模型组较对照组小鼠的肺泡灌洗液沉渣中总细胞数增多、中性粒细胞数增多及嗜酸性粒细胞数增多。1.3 OVA诱导的哮喘小鼠模型组较对照组小鼠血浆中IgE水平明显升高。1.4 HE染色显示哮喘小鼠模型组表现为上皮细胞的脱落、平滑肌束的增加以及基底膜增厚等气道重塑的表现。1.5 Masson染色显示哮喘小鼠模型组气道中胶原纤维含量明显增加。1.6免疫组织化学染色表明哮喘小鼠模型组气道中CTGF、FSTL1含量增加,且重塑相关标志物的含量也增加。1.7 Western blot显示哮喘小鼠模型组肺组织中CTGF、FSTL1及其受体DIP2A高表达,同时伴随重塑相关标志物的高表达。1.8 qRT-PCR显示哮喘小鼠模型组肺组织中CTGF、FSTL1及其受体DIP2A的mRNA高表达,同时伴随重塑相关标志物的高表达。2.CTGF通过FSTL1/DIP2A信号通路促进气道重塑。2.1 Western blot显示FSTL1重组蛋白刺激人肺成纤维细胞后其受体DIP2A蛋白高表达,同时伴随重塑相关标志物的高表达。与对照组相比,FSTL1重组蛋白刺激组FSTL1的受体DIP2A蛋白表达增高,同时重塑相关标志物的表达也增高(p<0.05),具有统计学差异。2.2 Western blot显示CTGF重组蛋白刺激人肺成纤维细胞后FSTL1及其受体DIP2A蛋白高表达,同时伴随重塑相关标志物的高表达。与对照组相比,CTGF重组蛋白刺激组FSTL1及其受体DIP2A蛋白表达增高,同时重塑相关标志物的表达也增高(p<0.05),具有统计学差异。2.3人肺成纤维细胞转染FSTL1 siRNA后给予CTGF重组蛋白处理,Western blot显示CTGF重组蛋白刺激阴性对照组人肺成纤维细胞后FSTL1、DIP2A及重塑相关标志物的表达较未经CTGF刺激的阴性对照组明显增高,且具有统计学差异(p<0.05)。CTGF重组蛋白刺激转染FSTL1 siRNA的人肺成纤维细胞后FSTL1、DIP2A及重塑相关标志物的表达较CTGF重组蛋白刺激阴性对照组显著减低,且有统计学差异(p<0.05)。CTGF重组蛋白刺激转染FSTL1 siRNA的人肺成纤维细胞后FSTL1、DIP2A及重塑相关标志物的表达较未经CTGF重组蛋白刺激转染FSTL1 siRNA的人肺成纤维细胞组表达无明显差异。(p>0.05)。提示予以CTGF重组蛋白刺激肺成纤维细胞后FSTL1、受体DIP2A、重塑标记物的表达均升高,阻断FSTL1后,FSTL1、受体DIP2A、重塑标记物的受到影响,未见升高。2.4人肺成纤维细胞转染DIP2A siRNA后给予CTGF重组蛋白处理,Western blot显示CTGF重组蛋白刺激阴性对照组人肺成纤维细胞后FSTL1、DIP2A及重塑相关标志物的表达较未经CTGF刺激的阴性对照组明显增高,且具有统计学差异(p<0.05)。CTGF重组蛋白刺激转染DIP2A siRNA的人肺成纤维细胞后DIP2A及重塑相关标志物的表达较CTGF重组蛋白刺激阴性对照组明显减低,且具有统计学差异(p<0.05),而FSTL1的表达基本不变,(p>0.05)。CTGF重组蛋白刺激转染DIP2A siRNA的人肺成纤维细胞后DIP2A及重塑相关标志物的表达较未经CTGF重组蛋白刺激转染DIP2A siRNA的人肺成纤维细胞组表达无明显差异,(p>0.05),而FSTL1的表达明显升高,(p<0.05)。2.5人肺成纤维细胞转染DIP2A siRNA后给予FSTL1重组蛋白处理,Western blot显示FSTL1重组蛋白刺激阴性对照组人肺成纤维细胞后重塑相关标志物的表达较未经FSTL1刺激的阴性对照组明显增高,且具有统计学差异(p<0.05),而CTGF的表达基本不变,(p>0.05)。FSTL1重组蛋白刺激转染DIP2A siRNA的人肺成纤维细胞后重塑相关标志物的表达较FSTL1重组蛋白刺激阴性对照组明显减低,且具有统计学差异(p<0.05),而CTGF的表达基本不变,(p>0.05)。FSTL1重组蛋白刺激转染DIP2A siRNA的人肺成纤维细胞后重塑相关标志物的表达较未经FSTL1重组蛋白刺激转染DIP2A siRNA的人肺成纤维细胞组表达无明显差异(p>0.05),而CTGF的表达基本不变,(p>0.05)。3.体内干预CTGF的表达后哮喘小鼠FSTL1和气道重塑受到影响。3.1给予乙酰甲胆碱雾化吸入后,WT+OVA组较WT组的气道阻力增加,WT+OVA+Dex组较WT+OVA组气道阻力减低,WT+OVA+CTGF组较WT+OVA组气道阻力增加。3.2 HE染色显示WT+OVA组小鼠模型组表现为上皮细胞脱落、平滑肌束增加以及基底膜增厚等气道重塑表现。WT+OVA+Dex组较WT+OVA组小鼠气道炎症及重塑减轻。WT+OVA+CTGF组较WT+OVA组小鼠气道炎症及重塑加重。3.3免疫组化显示WT+OVA组较WT组的FSTL1、重塑相关标志物增加,WT+OVA+Dex组较WT+OVA组的FSTL1、重塑相关标志物表达减低,WT+OVA+CTGF组较WT+OVA组的FSTL1、重塑相关标志物表达增加。3.4 Western blot显示WT+OVA组较WT组的FSTL1、重塑相关标志物表达增加,WT+OVA+Dex组较WT+OVA组的FSTL1、重塑相关标志物表达减低,WT+OVA+CTGF组较WT+OVA组的FSTL1、重塑相关标志物表达增加。3.5 qRT-PCR显示WT+OVA组较WT组的FSTL1、重塑相关标志物表达增加,WT+OVA+Dex组较WT+OVA组的FSTL1、重塑相关标志物表达减低,WT+OVA+CTGF组较WT+OVA组的FSTL1、重塑相关标志物表达增加。结论CTGF通过影响FSTL1/DIP2A信号通路促进哮喘气道重塑的发生。更多还原

【Abstract】 BackgroundBronchial asthma is a chronic airway inflammatory disease characterized by airway hyperresponsiveness,airway inflammation and airway remodeling.Current treatments for airway inflammation and airway hyperresponsiveness are effective in relieving and preventing symptoms in most patients,but some patients who experience persistent symptoms and progressive decline in lung function are described as irreversible or refractory asthma.Airway remodeling is one of the main features of asthma.This remodeling process is a key feature of severe asthma and is believed to play a key role in refractory or persistent asthma.Therefore,the research on the mechanism of airway remodeling and the development of biological agents for various components of airway remodeling are particularly necessary for asthma,especially refractory asthma.At present,animal models,vitro studies and some clinical studies have been put forward about the airway remodeling of the cellular and molecular pathways involved in the existing knowledge,which promotes the aim of airway remodeling of various components for the development of biological agents,in order to limit the airway remodeling in patients with asthma,to cure bronchial asthma to the one step closer.Connective tissue growth factor(CTGF)is an secreted peptide rich in cysteine.The expression of CTGF was significantly increased in the lung of ova-induced mice,and CTGF was positively correlated with MMP-9、TIMP-1 and smooth muscle layer thickness,suggesting that the up-regulation of CTGF was correlated with MMP-9,and CTGF might be involved in the pathogenesis of airway remodeling in asthma.Further studies have found that CTGF in plasma may be a potential biomarker for stable asthma when assessing the degree of persistent airway obstruction,and CTGF may be associated with airway remodeling in asthma.Follistatin-Like 1(Fstll)is an extracellular matrix glycoprotein belonging to the family of follicular statin proteins,which is a secretory protein.It plays an important role in embryo development,organ formation,tumor,inflammatory response and myocardial protection,and can improve endothelial function,promote vascular regeneration,and inhibit the proliferation and migration of vascular smooth muscle cells.Abnormal proliferation and migration of airway smooth muscle cells play an important role in airway remodeling in asthma and lead to airway hyperresponsiveness.Fstl1 has been shown to play an important role in promoting the proliferation and migration of human airway smooth muscle cells.In the lungs of patients with severe asthma,Fstll is highly expressed by macrophages,and Fstll knockout can inhibit smooth muscle cell migration,suggesting that Fstll may represent a new therapeutic target for airway remodeling in bronchial asthma.Disconnected(disco)interaction protein homologue 2 A(DIP2A)is a kind of cytoplasmic proteins.Immunocoprecipitation revealed the direct physical interaction between FSTL1 and DIP2A,which mediated the downstream action of FSTL1 as a receptor of FSTL1.Transforming growth factor beta(TGF-β)is a multifunctional protein that can affect the growth,differentiation,apoptosis and immune regulation of various cells.The level of TGF-β is increased in the airways of asthmatic patients.Many of the effects of TGF-β are mediated by CTGF and CTGF is one of the key fibrogenic factors related to TGF-β.TGF-β is the main central media of tissue fibrosis,Fstl1 is an important fibrinogen in the process of pulmonary tissue fibrosis,mainly through the TGF-βsignaling pathway to promote fibrosis.TGF-β can induce the formation of Fstll in lung fibroblasts and up-regulate the expression of Fstl1 in mouse pulmonary fibrosis.Many effects of TGF-β are mediated by CTGF.Previous studies have shown that both FSTL1 and CTGF can promote airway remodeling,but the specific mechanism of the action is still unclear.In this study,CTGF and FSTL1 were used as entry points to clarify the specific mechanism of airway remodeling and to provide clinical treatment ideas for the early prevention and treatment of asthma patients with fixed airflow limitation.ObjectivesTo explore whether CTGF promotes the occurrence of airway remodeling in asthma by affecting the FSTL1/DIP2A signaling pathway.Methods1.To clarify the role of CTGF and FSTL1 in remodeling allergic airway disease model in mice induced by OVA.1.1 female BALB/C mice at 6-8 weeks were given OVA to induce allergic airway disease model in mice.The allergic airway disease model was induced by intraperitoneal injection and local challenge by nasal drip inhalation.1.2 After the successful establishment of the model,the airway responsiveness of mice in each group was measured,and the airway resistance after methacholine atomization inhalation at different concentrations was recorded.1.3 After lung function monitoring,the bronchoalveolar lavage fluid,blood and lung tissue samples of mice in the asthma model group and the control group were collected.1.4 The bronchoalveolar lavage fluid of mice in each group was counted and classified.1.5 The concentration of IgE in peripheral blood of mice in each group was determined.1.6 HE staining was used to observe the characteristics of airway remodeling,including exfoliation of epithelial cells,increase of smooth muscle bundles and thickening of basement membrane.1.7 Masson staining was used to observe the expression of collagen fibers in airway mucosa of mice1.8 The expression of CTGF,FSTL1 and the expression of a-smooth muscle actin(a-SMA),fibronectin and collagen I were determined by immunohistochemistry.1.9 The expression of CTGF,FSTL1 and DIP2A,as well as remodeling related proteins were determined by Western blot.1.10 The expressions of CTGF,FSTL1 and its receptor DIP2A and remodeling related markers mRNA were detected by real-time quantitative polymerase chain reaction(qRT-PCR).2.The effect of CTGF on the FSTL1/DIP2A signaling pathway was observed at the cellular level.2.1 Human lung fibroblasts were cultured in vitro,and the expressions of DIP2A,a-SMA,fibronectin and collagen Ⅰ were detected after the stimulation of FSTL1 recombinant protein2.2 Human lung fibroblasts were cultured in vitro,and the expressions of FSTL1、DIP2A,a-SMA,fibronectin and collagen Ⅰ were detected after the stimulation of CTGF recombinant protein.2.3 Human lung fibroblasts were cultured in vitro,transfected with FSTL1 siRNA and treated with CTGF recombinant protein,and the expressions of FSTL1,DIP2A,a-SMA,fibronectin and collagen Ⅰ were detected2.4 Human lung fibroblasts were cultured in vitro,transfected with DIP2A siRNA,and treated with CTGF recombinant protein,and the expressions of FSTL1,DIP2A,a-SMA,fibronectin and collagen Ⅰ were detected.2.5 Human lung fibroblasts were cultured in vitro and transfected with DIP2A siRNA.After being stimulated by FSTL1 recombinant protein,the expressions of CTGF,a-SMA,fibronectin and type Ⅰ collagen were detected.3.Effects of intervention of CTGF expression on FSTL1 and airway remodeling in asthmatic mice in vivo.3.1 female BALB/C mice of 8 weeks old were randomly divided into WT group,WT+OVA group,WT+OVA+Dex group and WT+OVA+CTGF group.The OVA asthma mouse model was established in WT+OVA group by the method described in the first part.WT+OVA+ Dex group was injected intraperitoneally with dexamethasone.WT+OVA+CTGF was intranasally dripped with CTGF recombinant protein.Both of them were on the basis of WT+OVA group model preparation.3.2 After the successful establishment of the model,the airway responsiveness of mice in each group was measured,and the airway resistance after methacholine atomization inhalation at different concentrations was recorded.3.3 After pulmonary function monitoring,the mice were killed and the lung tissue samples of each group were collected.3.4 HE staining was used to observe the characteristics of airway remodeling in each group,including exfoliation of epithelial cells,increase of smooth muscle bundles and thickening of basement membrane.3.5 The expression of FSTL1 and remodeling related markers a-SMA,fibronectin and type I collagen were detected by immunohistochemistry.3.6 The expression of FSTL1 and remodeling related proteins were determined by Western blot.3.7 The expressions of FSTL1 and remodeling related markers mRNA were detected by real-time quantitative polymerase chain reaction(qRT-PCR).Results1.Airway remodeling in asthmatic mice was increased,accompanied by increased expression of CTGF and FSTL1.1.1 The airway responsiveness of mice with allergic airway disease induced by OVA in the model group was higher than that in the control group.1.2 Compared with the control group,the number of total cells,neutrophils and eosinophils in the bronchoalveolar lavage fluid(BALF)of OVA-induced mice increased.1.3 The level of plasma IgE in OVA-induced asthmatic mice was significantly higher than that in the control group.1.4 HE staining showed that airway remodeling in the asthmatic mouse model group was characterized by epithelial cell shedding,increased smooth muscle tracts and thickened basement membrane1.5 Masson staining showed a significant increase in collagen fibers in the airway tissues of the asthmatic mouse model group1.6 Immunohistochemistry showed high expression of CTGF and FSTL1 in the airway mucosa of asthmatic mouse model,accompanied by high expression of remodeling related proteins a-SMA,fibronectin and collagen Ⅰ1.7 Western blot showed high expression of CTGF,FSTL1 and its receptor DIP2A in lung tissues of asthmatic mice,accompanied by high expression of remodeling related proteins a-SMA,fibronectin and collagen Ⅰ1.8 qRT-PCR showed high expression of CTGF,FSTL1 and their receptor DIP2A mRNA in the lung tissue of asthmatic mice,accompanied by high expression of remodeling-related proteins a-SMA,fibronectin and type Ⅰ collagen mRNA2.CTGF promotes airway remodeling through the FSTL1/DIP2A signaling pathway.2.1 Western blot showed that human lung fibroblasts stimulated by recombinant FSTL1 protein had high expression of its receptor DIP2A protein,accompanied by high expression of remodeling-related proteins.Compared with the control group,the expression of DIP2A protein of FSTL1 receptor and the expression of remodeling-related proteins in FSTL1 recombinant protein group were significantly higher than those in control group(p<0.05).2.2 Western blot showed that FSTL1 and its receptor DIP2A protein were highly expressed in human lung fibroblasts stimulated by CTGF recombinant protein,accompanied by high expression of remodeling-related proteins.Compared with the control group,the expression of FSTL1 and its receptor DIP2A protein and remodeling-related proteins in the CTGF recombinant protein group were significantly higher than those in the control group(p<0.05).2.3 After human lung fibroblasts were transfected with FSTL1 siRNA and treated with CTGF recombinant protein,Western blot showed that the expressions of FSTL1,DIP2A and remodeling-related proteins in human lung fibroblasts in the negative control group stimulated by CTGF recombinant protein were significantly higher than those in the negative control group without CTGF stimulation.The expressions of FSTL1,DIP2A and remodeling related proteins in FSTL1 siRNA transfected human lung fibroblasts stimulated by CTGF recombinant protein were significantly lower than those in the negative control group stimulated by CTGF recombinant protein.The expressions of FSTL1,DIP2A and remodeling related proteins in human lung fibroblasts transfected with CTGF recombinant protein were not significantly different from those in human lung fibroblasts transfected with FSTL1 siRNA without CTGF recombinant protein stimulation.(P>0.05).It is suggested that the expression of FSTL1,receptor DIP2A and remodeling markers in pulmonary fibroblasts stimulated by recombinant CTGF protein increased.After blocking FSTL1,the expressions of FSTL1,receptor DIP2A and remodeling markers were affected and did not increase.2.4 After human lung fibroblasts were transfected with DIP2A siRNA and treated with CTGF recombinant protein,Western blot showed that the expressions of FSTL1,DIP2A and remodeling-related proteins in human lung fibroblasts in the negative control group stimulated by CTGF recombinant protein were significantly higher than those in the negative control group without CTGF stimulation.The expressions of DIP2A and remodeling related proteins in DIP2A siRNA transfected human lung fibroblasts stimulated by CTGF recombinant protein were significantly lower than those in the negative control group stimulated by CTGF recombinant protein,while the expression of FSTL1 was almost unchanged.The expression of DIP2A and remodeling related proteins in human lung fibroblasts transfected with DIP2A siRNA stimulated by recombinant CTGF protein were not significantly different from those in human lung fibroblasts transfected with DIP2A siRNA without CTGF recombinant protein(p>0.05),while the expression of FSTL1 was significantly increased in human lung fibroblasts transfected with DIP2A siRNA2.5 Human lung fibroblasts were treated with FSTL1 recombinant protein after transfection of DIP2A siRNA,Western blot showed that the expression of remodeling-related proteins in human lung fibroblasts stimulated by FSTL1 recombinant protein was significantly higher than that in the negative control group without FSTL1 stimulation,while the expression of CTGF was basically unchanged.The expression of remodeling-related proteins in DIP2A siRNA-transfected human lung fibroblasts stimulated by recombinant FSTL1 protein was significantly lower than that in negative control group stimulated by FSTL1 recombinant protein,while the expression of CTGF remained unchanged.The expression of remodeling-related proteins in DIP2A siRNA-transfected human lung fibroblasts stimulated by recombinant FSTL1 protein was not significantly different from that in DIP2A siRNA-transfected human lung fibroblasts without FSTL1 recombinant protein stimulation(p>0.05),while the expression of CTGF was almost unchanged in human lung fibroblasts transfected with DIP2A siRNA.3.FSTL1 and airway remodeling in asthmatic mice were affected by in vivo after intervention of CTGF expression.3.1 After aerosol inhalation of methacholine,the airway resistance of WT+OVA group was higher than that of WT group,the airway resistance of WT+OVA+Dex group was lower than that of WT+OVA group,and the airway resistance of WT+OVA+CTGF group was higher than that of WT+OVA group.3.2 HE staining showed that the WT+OVA model group showed airway remodeling such as exfoliation of epithelial cells,increase of smooth muscle bundles and thickening of basement membrane.The airway inflammation and remodeling in WT+OVA+Dex group were less than those in WT+OVA group.The airway inflammation and remodeling in WT+OVA+CTGF group were more serious than those in WT+OVA group3.3 Immunohistochemistry showed that the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA group was higher than that in WT group,the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA+Dex group was lower than that in WT+OVA group,and the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA+CTGF group was higher than that in WT+OVA group.3.4 Western blot showed that the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA group was higher than that in WT group,the expression of FSTL1,Collagen I,Fibronectin and a-SMA in WT+OVA+Dex group was lower than that in WT+OVA group,and the expression of FSTL1,Collagen I,Fibronectin and a-SMA in WT+OVA+CTGF group was higher than that in WT+OVA group.3.5 qRT-PCR showed that the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA group was higher than that in WT group,the expression of FSTL1,Collagen I,Fibronectin and a-SMA in WT+OVA+Dex group was lower than that in WT+OVA group,and the expression of FSTL1,Collagen Ⅰ,Fibronectin and a-SMA in WT+OVA+CTGF group was higher than that in WT+OVA group.ConclusionCTGF promotes airway remodeling in asthma by affecting FSTL1/DIP2A signaling pathway.更多还原