ADAM33表达量与气道平滑肌细胞生物力学属性的关系研究

发布时间：2018-09-14 19:20

【摘要】：哮喘(Asthma)是一种常见的慢性呼吸道系统疾病，涉及环境因素与遗传因素之间的复杂相互作用，是公认的医学难题，严重威胁着人类的生命健康。然而其生理病理机制至今还没有得到完全地解释。有研究表明，哮喘的病理机制中包括气道重塑(airway remodeling)，气道炎症(Airway inflammation)以及气道高反应性(Airway hyperresponsiveness，AHR)等。目前国际上公认的哮喘可能的发病机制是，气道平滑肌细胞(Airway smooth muscle cell, ASMC)自身生物力学属性发生改变，当受到外界理化因素刺激时，其反应性增强导致气道高反应性与气道狭窄以及随之而来的呼吸功能障碍。因此气道平滑肌生物力学属性变异作为此途径的终极因素成为研究哮喘生理病理机制的重点。近年来，通过定位克隆和全基因组筛查技术确定了去整合素碱金属蛋白酶33(A disintegrin and metalloproteinase33, ADAM33)是一种哮喘易感基因，且发现ADAM33基因与哮喘疾病特征的气道炎症和组织重构具有一定联系。然而与气道炎症同等重要的哮喘气道病理变化是源于ASMC生物力学属性改变所导致的AHR，且已有研究表明ADAM33在哮喘患者气道组织的间充质层包括成纤维层、基底膜和平滑肌层上的表达水平比正常人高。由此认为在哮喘发生发展过程中ASMC的ADAM33蛋白表达与ASMC生物力学属性是否具有一定的相关性、以及ADAM33蛋白的表达是否参与调控ASMC力学特性。为了解决这个问题，本课题采用卵清蛋白(OVA)致敏新生雄性SD大鼠构建哮喘动物模型，在此基础上分离纯化ASMC在体外进行原代培养，研究哮喘病理气道中ASMC的ADAM33蛋白表达水平，及其与ASMC生物力学行为之间的相关性，同时检测在体外调控ADAM33蛋白表达后ASMC细胞生物力学行为的响应，研究两者之间的关系。主要研究内容及结果如下： ①研究了不同程度OVA致敏雄性SD大鼠哮喘模型ASMC的ADAM33蛋白表达水平。首先将新生雄性SD大鼠随机分为4组，哮喘组在第1和第7天时注射含1%OVA和10%Al(OH)3的混合盐溶液，第15天起用1%OVA每周雾化3次，分别持续4、6和12周以建立不同程度慢性OVA致敏动物，正常对照组动物用相同的实验方案采取同等剂量的生理盐水处理。从动物模型中分离、获取ASMC并在体外进行原代培养，细胞传代至2-5代用于各项实验。当细胞长至90%融合时，提取总蛋白，将等体积的蛋白溶液加入到10%SDS-PAGE胶中，随后用免疫印记方法(western blot)检测细胞中ADAM33蛋白表达水平，实验结果表明OVA致敏组ASMC中ADAM33蛋白表达水平显著高于对照组(p0.001)，增加水平在OVA致敏4周时达到最大，致敏6和12周表达水平渐次； ②研究了正常与OVA致敏来源的ASMC生物力学属性。从气道的功能来看，ASMC的力学属性被认为是决定哮喘病症之一气道高反应的最终途径。因此本研究综合采取光学磁粒扭转细胞测量仪(Optical Magnetic Twisting Cytometry,OMTC)、傅立叶变换牵引力显微术(Fourier Transform Traction Microscopy,FTTM)、Transwell小室法以及免疫荧光染色技术检测包括细胞刚度和收缩性、细胞牵引力、细胞迁移以及细胞骨架结构蛋白表达在内的细胞力学属性。实验结果表明，与对照组相比OVA致敏组ASMC细胞刚度增加，致敏4周时具有显著性差异(p0.05)，随后渐次，然而各组细胞对KCl激动剂的响应没有显著性差异。同样，OVA致敏组中ASMC牵引力提高，尤其在OVA致敏4周时具有显著性差异(p0.05)，随后随着致敏时间的增加有所降低。OVA致敏组ASMC的迁移能力增加，同样的致敏4周时达到最大具有显著性差异(p0.001)。致敏组ASMC中包括F-actin和Vinculin的细胞骨架结构蛋白的表达较对照组有所提高，特别是致敏4周时，Vinculin的表达具有显著性差异（p0.001）。 ③分析了ADAM33蛋白表达水平与ASMC力学属性之间的相关性。实验结果表明，ASMC的ADAM33蛋白表达的变化和ASMC力学属性的改变对OVA致敏具有相同的响应趋势，暗示两者之间似乎具有一定的相关性。通过相关性分析发现ASMC中ADAM33蛋白的表达水平与细胞刚度、细胞牵引力、细胞迁移以及包括F-actin和Vinculin的细胞骨架结构蛋白表达在内的细胞力学属性具有很强的正相关性(Pearson相关系数Cp：分别是0.864，0.716，0.67，0.662，0.774，如表3.1所示)。 ④研究了调控ADAM33蛋白表达后ASMC细胞力学属性的响应。用上述实验中发现的蛋白表达与力学属性改变最大的OVA致敏4周的ASMC，通过慢病毒转染的办法构建沉默ADAM33蛋白表达的ASMC组，然后检测细胞的力学属性响应。我们的实验结果发现与无处理对照组(4weeks)以及慢病毒转染对照组(GFP)相比，沉默组(pLVT453)细胞的刚度与细胞牵引力都显著下降(p0.05)。这些结果表明，ADAM33蛋白与ASMC生物力学属性具有很强的正相关性，同时与ASMC力学属性的改变有着直接的联系，并进而在气道高反应性这一哮喘病理因素中发挥关键作用。通过研究ADAM33的表达对ASMC生物力学行为的影响及其内在调控机理，有助于进一步揭示支气管哮喘的发病机制，，为探索更好的哮喘病治疗靶点提供科学依据。
[Abstract]:Asthma is a common chronic respiratory disease, involving the complex interaction between environmental factors and genetic factors. It is a recognized medical problem, seriously threatening human life and health. However, its physiological and pathological mechanisms have not been fully explained. Studies have shown that the pathogenesis of asthma includes airway. Airway remodeling, airway inflammation, and airway hyperresponsiveness (AHR) have been widely recognized as possible pathogenesis of asthma. The enhancement of airway responsiveness leads to airway hyperresponsiveness, airway stenosis and subsequent respiratory dysfunction. Therefore, the variation of airway smooth muscle biomechanical properties as the ultimate factor in this pathway has become the focus of the study on the physiological and pathological mechanism of asthma.
In recent years, disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptible gene for asthma by localized cloning and genome-wide screening techniques, and it has been found that ADAM33 gene is associated with airway inflammation and tissue remodeling characterized by asthma. The pathological changes of airway in asthma are caused by the changes of biomechanical properties of ASMC, and the expression of ADAM33 in the airway mesenchymal layer including fibroblast layer, basement membrane and smooth muscle layer of asthmatic patients is higher than that of normal people. To solve this problem, we used ovalbumin (OVA) sensitized neonatal male SD rats to construct an asthmatic animal model, and then isolated and purified ASMC for primary culture in vitro to study asthmatic pathological qi. The expression level of ADAM33 protein in ASMC and its correlation with the biomechanical behavior of ASMC were studied. The response of ASMC cells to the biomechanical behavior of ADAM33 protein was also detected in vitro.
(1) The expression of ADAM33 protein in ASMC of male SD rats sensitized by OVA was studied. Firstly, neonatal male SD rats were randomly divided into 4 groups. Asthma group was injected with mixed saline solution containing 1% OVA and 10% Al (OH) 3 on the 1st and 7th day, and 1% OVA was atomized three times a week on the 15th day for 4, 6 and 12 weeks respectively to establish different degrees of chronic OVA. VA-sensitized animals and normal control animals were treated with the same dosage of normal saline. ASMCs were isolated from animal models and cultured in vitro. Cells were subcultured to 2-5 generations for various experiments. When the cells grew to 90% fusion, total proteins were extracted and the same volume of protein solution was added to 10% SDS-PAGE. The expression of ADAM33 protein in ASMC of OVA sensitized group was significantly higher than that of control group (p0.001). The expression level of ADAM33 protein in ASMC of OVA sensitized group reached the maximum at 4 weeks, and gradually increased at 6 and 12 weeks.
(2) The biomechanical properties of ASMC from normal and OVA sensitized sources were studied. The mechanical properties of ASMC were considered to be the ultimate way to determine airway hyperresponsiveness in asthmatic patients. Fourier Transform Traction Microscopy (FTTM), Transwell chamber assay and immunofluorescence staining were used to detect the cellular mechanical properties including cell stiffness and contractility, cell traction, cell migration and cytoskeletal structural protein expression. Similarly, the traction of ASMC in OVA sensitized group increased significantly, especially in OVA sensitized 4 weeks (p0.05), and then decreased with the increase of sensitization time. The migration ability of ASMC in OVA sensitized group decreased. The expression of cytoskeleton structural protein including F-actin and Vinculin in ASMC of the sensitized group was higher than that of the control group. Especially, the expression of Vinculin in ASMC of the sensitized group was significantly higher than that of the control group at 4 weeks (p0.001).
(3) The correlation between ADAM33 protein expression and mechanical properties of ASMC was analyzed. The results showed that the changes of ADAM33 protein expression and mechanical properties of ASMC had the same response trend to OVA sensitization, suggesting that there was a certain correlation between them. Pearson correlation coefficients Cp: 0.864, 0.716, 0.67, 0.662, 0.774 respectively, as shown in Table 3.1.
(4) The response of mechanical properties of ASMC cells to ADAM33 protein expression was studied. ASMC cells were sensitized by OVA with the greatest change of protein expression and mechanical properties found in the above experiments for 4 weeks. The ASMC group silenced ADAM33 protein expression was constructed by lentivirus transfection, and then the mechanical properties of ASMC cells were detected. Compared with the untreated control group (4weeks) and lentivirus transfection control group (GFP), the stiffness and traction of cells in the silent group (pLVT453) were significantly decreased (p0.05).
These results indicate that ADAM33 protein has a strong positive correlation with the biomechanical properties of ASMC, and is directly related to the changes of mechanical properties of ASMC, and plays a key role in airway hyperresponsiveness, a pathological factor of asthma. It is helpful to further reveal the pathogenesis of bronchial asthma and provide scientific basis for exploring better therapeutic targets of asthma.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R562.25

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