【作者】 谢敏；

【导师】 徐永健；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 支气管哮喘是一种世界范围内广泛发病的常见病、多发病,其发病机制至今尚未完全阐明。近年的研究发现,气道重建是慢性持续性哮喘和重度哮喘患者难以得到有效治疗的重要原因之一,相关机制正成为目前国内外的研究热点。气道平滑肌细胞作为气道重建中的主要分子,参与了气道重建的始动及发展整个过程。它作为结构细胞不仅通过自身的肥大与增生直接参与气道壁的增厚,而且通过表型转化、迁移功能的改变以及分泌炎症因子和细胞外基质等促进气道重建的发展。然而有关哮喘支气管平滑肌表型转化、迁移功能和分泌功能改变的发生机制目前仍不明确。细胞外信号调节激酶属于有丝分裂原激活蛋白激酶家族,它是细胞内重要的信号传递分子,参与细胞生长、发育、增殖、分化等多种生理病理过程,它有多种亚型,在平滑肌中主要有ERK1、ERK2(ERK1/2)两种,有研究表明它在哮喘气道慢性炎症和气道高反应性中起到了重要的调控作用,但是有关ERK信号通道在哮喘支气管平滑肌表型转化、迁移和分泌功能改变中作用的研究国内外尚少见报道。本研究旨在通过大鼠慢性哮喘模型和哮喘血清被动致敏人支气管平滑肌细胞探讨ERK1/2信号通道在哮喘支气管平滑肌细胞表型转化、迁移和分泌功能改变中的调控作用,为进一步阐明哮喘气道重建的发病机制提供实验依据。方法:应用卵清蛋白雾化和激发的方法制备大鼠慢性哮喘模型,应用哮喘患者血清被动致敏人支气管平滑肌细胞,气道病理切片检测气道改变,免疫组织化学、免疫荧光细胞化学、western blot和RT-PCR等方法检测总ERK1/2、磷酸化ERK1/2和平滑肌标志蛋白(alpha-actin和骨桥蛋白)在支气管平滑肌细胞中的表达,透射电镜下观察支气管平滑肌细胞超微结构的改变,平面迁移实验和跨膜迁移实验检测支气管平滑肌细胞迁移能力的改变,ELISA方法检测支气管平滑肌细胞分泌功能的变化。同时,还运用了ERK1/2信号通道的激动剂表皮生长因子、抑制剂PD98059和ERK1/2反义寡核苷酸对ERK1/2信号通道进行了干预。结果:1.在慢性哮喘模型大鼠气道中ERK1/2的活性和在蛋白质和核酸水平上的表达均明显增强(P<0.01),且P-ERK的表达与气道管壁厚度显著正相关。2.慢性哮喘模型大鼠支气管平滑肌细胞超微结构出现大量合成细胞器,同时平滑肌收缩标志蛋白(alpha-actin)的表达下降,合成型标志蛋白(osteopontin)的表达上升(P<0.01)。ERK1/2信号通道激动剂表皮生长因子(epidermal growth factor, EGF)促进了这一转变,而抑制剂PD98059部分抑制了平滑肌细胞表型的转化。3.慢性哮喘模型大鼠支气管平滑肌细胞的平面迁移和跨膜迁移能力都较正常对照大鼠有明显增强,10μmol/L的PD98059即明显抑制慢性哮喘支气管平滑肌细胞的迁移,而EGF有显著的促进作用,ERK1/2反义寡核苷酸转染能够有效抑制细胞的迁移能力(P<0.01)。4.慢性哮喘大鼠支气管平滑肌细胞较正常对照分泌转化生长因子、血管内皮生长因子、结缔组织生长因子、RANTES、嗜酸细胞活化趋化因子、纤维结合蛋白和Ⅰ型胶原蛋白的水平均有明显升高(P<0.01)。EGF能够促进正常大鼠BSMC和慢性哮喘大鼠的分泌,但慢性哮喘组的水平要显著高于正常对照,而PD98059降低了慢性哮喘大鼠支气管平滑肌细胞的分泌水平(P<0.01),ERK1/2反义寡核苷酸能够有效的抑制慢性哮喘大鼠支气管平滑肌细胞的分泌(P<0.01)。5.经鼻吸入ERK1/2反义寡核苷酸下调ERK1/2在慢性哮喘气道平滑肌的表达,并抑制了慢性哮喘模型大鼠气道壁的增厚,阻止了支气管平滑肌细胞的表型转化(P<0.01)。6.哮喘血清被动致敏的人支气管平滑肌细胞向合成细胞型进一步转化,EGF促进了被动致敏平滑肌细胞的表型转化,PD98059降低合成型标志蛋白的表达,升高收缩型标志蛋白的水平(P<0.05)。ERK1/2反义寡核苷酸能够有效抑制被动致敏人支气管平滑肌细胞的表型转化(P<0.05)。7.哮喘血清被动致敏的人支气管平滑肌细胞的迁移能力较正常血清对照有明显增强,EGF促进了被动致敏平滑肌细胞的迁移,而PD98059抑制了细胞的迁移能力(P<0.05),ERK1/2反义寡核苷酸转染能够有效的抑制被动致敏人支气管平滑肌细胞的迁移能力的增强(P<0.05)。8.哮喘血清被动致敏的人支气管平滑肌细胞分泌生长因子、炎性因子和细胞外基质的能力较正常血清对照细胞有明显增强,EGF促进了被动致敏平滑肌细胞的分泌,而PD98059降低细胞的分泌水平(P<0.05),ERK1/2反义寡核苷酸转染能够有效的抑制被动致敏人支气管平滑肌细胞的分泌能力(P<0.05)。结论:ERK1/2信号通道参与了慢性哮喘模型大鼠支气管平滑肌细胞和哮喘患者血清被动致敏人支气管平滑肌细胞表型转化、迁移和分泌功能变化的调控,提示ERK信号通道在慢性哮喘气道重建发病机制中可能具有重要作用。更多还原

【Abstract】 Background:Asthma is a chronic inflammatory disorder of airway that is highly prevalent in world and its pathogenesis remains completely uncertain. Despite an increased use of medications that suppress airway inflammation and repress contraction of smooth muscle, some patients with chronic and severe asthma still hardly get effective treatment due to the development of airway remodeling and subsequent poorly reversible airway obstruction. Of the structural changes in the airway wall remodeling process, bronchial smooth muscle cells (BSMCs) are perhaps the most important component. Recent reports show that BSMCs not only contribute to airway remodeling by its increased content, but also, bronchial smooth muscle cells in chronic asthma are capable to undergo phenotypical transition, enabling them to subserve contractile, proliferative, migratory and secretory functional responses that account for airway remodeling and persistent hyperresponsiveness.However, despite consensus toward the importance of BSMCs regarding to the phenotypic plastic, migration and secretion, there is relatively little understanding of the cellular and molecular mechanisms underlying these changes seen in the asthmatic BSMCs. Extracellular signal-regulated kinase1/2 (ERK1/2) signaling pathway is one of the most important signalling pathways that modulate asthma in the chronic airway inflammation and hyperresponsiveness. Nevertheless, there are few reports about the role of ERK1/2 signaling pathway in the regulation of asthmatic BSMCs on their phenotypic modulation and migration. So the aim of this study is to profile the impact of ERK1/2 signaling pathway on the phenotypic transition, migration and secretion of BSMCs in chronic asthmatic model of rats, as well as in passively sensitized human BSMCs by asthmatic serum.Method:Wistar rats underwent OVA intraperitoneal injection and eight weeks inhalation for chronic asthma model. Human BSMCs were sensitized by asthmatic serum. Airway reactivity and relative count of eosinophil in alveolar lavage fluid were measured, and histological sections were investigated for morphometric analysis. The expressions of ERK1/2 and P-ERK1/2 at both protein and mRNA levels were detected by western blot and RT-PCR. Phenotype of BSMCs was established under electron microscrope, also with analysis of phenotypic markers (sm-α-actin for contractile and osteopontin for synthetic) in both protein and mRNA levels by western blot or RT-PCR, respectively. Migration of BSMCs was measured by plate test and transmembrane test. Secretion of BSMCs was measured by ELISA and western blot.Results:1. ERK1/2 was markedly increased in its expression and activity in the rat model of chronic asthma, and the level of P-ERK1/2 closely related to airway thickness.2. The phenotype of BSMCs in chronic asthmatic rats switched from a contractile type to a synthetic type with plentiful synthetic organelles gathering around the nucleus and altered expressions of phenotypic markers. EGF urged the over-synthetic function of BSMCs, while MEK inhibitor PD98059 was capable to reverse the phenotypic change of BSMCs.3. BSMCs of chronic asthmatic rats possessed increased migratory ability. EGF promoted the migration of BSMCs, MEK inhibitor PD98059 was capable to inhibit the change of BSMCs, and ERK1/2 antisense ODN restrained the migration ability of BSMCs.4. BSMCs of chronic asthmatic rats secreted much more growth factors (TGF-β1 , VEGF and CTGF), cytokines (RANTES and EOTAXIN) and extracellular matrix (Fibronectin and CollangenⅠ) than that of normal controls. EGF stimulated the secretion of both two groups, but the response of chronic asthmatic group was more intense. Both PD98059 and antisense oligonucleotide were able to suppress the secretion of BSMCs in chronic ashmatic rats, but antisense oligonucleotide reduced the level of RANTES nearly to that of normal controls, while PD98059 couldn’t.5. Local intranasal administration of ERK1/2 antisense oligonucleotides to chronic asthmatic rats led to DNA uptake in lung cells associated with a reduction of intracellular ERK1/2 expression. Such intrapulmonary blockade of ERK1/2 expression caused an abrogation of the increase in the thickness of airway as well as in that of smooth muscle layer. Furthermore, treatment with antisense but not nonsense oligonucleotides inhibited the phenotypic transition of BSMCs in the rat model of chronic asthma.6. Passively sensitized human BSMCs changed further toward the synthetic phenotype as compared to the controls. EGF pushed the phenotypic transition of human BSMCs, and PD98059 inhibited partly the change in human BSMCs. Meanwhile, ERK1/2 antisense ODN was capable to reverse the phenotypic change of passively sensitized human BSMCs.7. The migratory ability was increased in passively sensitized BSMCs as compared to the controls. EGF promoted the migration of human BSMCs, MEK inhibitor PD98059 was capable to inhibit the change of BSMCs, and ERK1/2 antisense ODN suppressed the migration ability of BSMCs.8. Passively sensitized human BSMCs secreted much more growth factors, cytokines and extracellular matrix than that of controls. EGF stimulated the secretion of human BSMCs, and PD98059 was able to suppress the secretion of human BSMCs. While ERK1/2 antisense oligonucleotide induced a dramatic reduction of the secreted factors to levels comparable to that of normal controls.Conclusion:ERK1/2 signalling pathway play an important role on the phenotypic modulation, migration and secretion of bronchial smooth muscle cells in the rat model of chronic asthma, as well as in the model of human BSMCs passively sensitized by asthmatic serum.更多还原