哮喘易感基因ADAM33对气道平滑肌细胞力学行为的影响

发布时间：2018-04-27 17:50

本文选题：哮喘 + 去整合素金属蛋白酶　；参考：《重庆大学》2012年博士论文

【摘要】：哮喘病是一种常见的呼吸道慢性炎症疾病，是指支气管在高反应状态下，由变应原或其他因素引起的气道狭窄的疾病，主要症状有胸闷、咳嗽、呼吸困难等。气道平滑肌细胞龴Airway Smooth Muscle Cell，ASMC㖞是呼吸道的主要组成部分之一，具有重要的生理作用。当气道平滑肌在受到外来因子的刺激时会发生气道过度收缩反应（Airway hyperrespinsiveness，AHR），临床表现为患者气道过度缩窄和发作性呼吸障碍，这是导致哮喘病成为高危疾病的最终原因所在。虽然在哮喘病的研究当中，对ASMC收缩特性的认识已经有一个多世纪，但是关于气道平滑肌病理变化的机制却没有研究透彻。气道平滑肌是控制气管管径的效应器，其病理性收缩成为气道阻塞和呼吸困难的最终因素，它在决定哮喘病治疗方法的有效性方面起到关键的作用，普遍认为其病理性收缩是哮喘难治的重要的病理基础。去整合素碱金属蛋白酶33（A disintegrin and metalloprotease33，ADAM33）基因已被证实是一个哮喘易感基因，且在大部分哮喘患者的气道平滑肌和基底膜上其表达水平较健康受试者要高，说明ADAM33可能参与ASMC力学环境的调控，进而与哮喘病人ASMC力学特性异常及气道重构有关。然而，至今还没有直接的证据证明ASMC生物力学行为与其ADAM33表达之间的关系。因此，本文采用分子生物学方法调控ASMC的ADAM33表达，同时采用细胞生物力学方法测量超表达和沉默ADAM33的ASMC的力学行为，以揭示二者之间的直接关系。本文的主要研究内容和研究结果如下： ①ADAM33的表达水平随着SD大鼠雾化周数的增加而显著性升高。首先，利用氢氧化铝作为佐剂，卵清蛋白（OVA）致敏并激发哮喘，建立SD(SpragueDawley)大鼠哮喘模型。每周隔天OVA雾化3次，每次30分钟，共雾化12周，每2周取大鼠肺组织。通过对肺功能（Penh）的测定及气道组织HE(Hematoxylin-Eosin staining)染色观察结果表明我们已成功构建大鼠哮喘模型。接着，利用定量RT-PCR和Western blot技术检测ADAM33在OVA雾化不同周数的SD大鼠哮喘平滑肌细胞中的表达，结果显示ADAM33的表达水平随着OVA雾化周数的增加而显著性升高，且定量RT-PCR与Western blot的实验结果基本一致。从而说明ADAM33的表达水平与哮喘的严重程度呈正相关，该结果与Lee等在哮喘病人中的研究发现相符合，更近一步证明我们实验中所构建的SD大鼠哮喘模型能真实地模拟哮喘病。 ②获得ADAM33超表达和沉默的ASMC。首先，构建ADAM33基因超表达和干扰慢病毒重组载体，然后进行慢病毒转染，以GFP空载及未转染细胞作为对照。在荧光显微镜下观察到重组慢病毒转染正常SD大鼠ASMC，24h后GFP表达逐渐增强，至培养72h时GFP表达最强到高峰，并发现当MOI(Multiplicity ofInfection)=100时，慢病毒感染ASMC的效率为95%。定量RT-PCR检测结果显示，超表达组ADAM33基因表达水平是正常对照组的8倍（P＜0.01），沉默组中pLVT453沉默效率最高达到75%（P＜0.01）；同时，Western blot测定各组细胞的ADAM33蛋白表达水平结果表明，超表达组细胞内ADAM33蛋白表达较两组对照明显增高（P＜0.01），而沉默组表达明显降低（P＜0.01），ADAM33蛋白在两组对照组中的表达量无明显差别。本实验成功获得了稳定的ADAM33超表达和沉默ASMC，且重组慢病毒转染明显上调或抑制了ASMC中ADAM33基因mRNA和蛋白的表达。 ③ADAM33基因表达水平的变化改变了ASMC的迁移能力和增殖能力。采用MTT法观察不同4组细胞增殖情况，发现超表达组pLV-ADAM33的细胞增殖能力与空白对照组相比没有显著性差异；而沉默组pMa-ADAM33相对于对空白照组细胞增殖能力明显降低（P＜0.05）。GFP对照组的细胞增殖能力显著低于未转染对照组（P＜0.01），，由此我们可以看出慢病毒转染对细胞增殖能力有抑制作用，虽然上调ADAM33对细胞增殖能力对于未转染对照组来说无差异，但是相对GFP对照组的细胞增殖能力显著增强（P＜0.01）。划痕法(Wound healing)检测细胞横向迁移能力的结果显示，在12h时，4组细胞迁移无明显差异。但是在24h时，超表达组pLV-ADAM33的横向迁移能力与两组对照相比明显增强（P＜0.01）（P＜0.05）；而沉默组pMa-ADAM33的细胞显著降低（P＜0.01），该结果表明通过特异性改变ADAM33基因的表达，能够影响ASMC横向迁移能力。 ④ADAM33基因能调控ASMC的细胞基础刚度、收缩力及牵张力等细胞力学特性。运用光学捕捉（Optical trapping）与磁力扭转细胞测量术（Magnetic TwistingCytometry）和傅里叶变换牵引力显微术（Fourier Transform Traction Microscopy，FTTM）检测由慢病毒转染所获得的稳定超表达和干扰ADAM33的ASMC的生物力学特性。研究结果显示：1)超表达ADAM33的ASMC组pLV-ADAM33的细胞基础刚度、KCL激动剂处理后的收缩力、牵张力显著高于对照组，而ADAM33沉默的细胞组其上述参数明显低于对照。2)共聚焦免疫荧光结果显示ADAM33和黏着斑蛋白vinculin存在共定位关系，并且还有部分ADAM33有沿着应力纤维分布的情况，说明ADAM33可能调控细胞骨架结构和粘着斑的形成。3)通过定量RT-PCR检测结果发现，ADMA33基因表达水平的改变能调控细胞力学相关基因Calponin和Integrin-β1的表达。从以上实验结果，我们推测：调控ADAM33的表达直接影响到ASMCs的生物力学行为，提示ASMCs的生物力学行为与其ADAM33的表达存在直接的联系。因此，哮喘患者中ADAM33基因表达及修复的异常有可能在气道重塑和气道收缩高反应性等方面扮演决定性的角色，这对于深入理解哮喘的发病机制，特别是气道功能变换的内在调控机制，从而探寻有效的哮喘治疗靶点和药物具有深远的意义。
[Abstract]:Asthma is a common chronic inflammatory disease of the respiratory tract. It refers to the bronchial stenosis caused by allergen or other factors in the high response state. The main symptoms are chest tightness, cough, and dyspnea. Airway smooth muscle cells? Airway Smooth Muscle Cell, ASMC? Is one of the main parts of the respiratory tract. When the airway smooth muscle is stimulated by foreign factors, the airway overcontraction (Airway hyperrespinsiveness, AHR) may occur. The clinical manifestation is the patient's airway overcontraction and paroxysmal breathing disorder, which is the ultimate cause of asthma to become a high-risk disease. Although in asthma studies, A The understanding of the contractile characteristics of SMC has been over a century, but the mechanism of the pathological changes of the airway smooth muscle is not thoroughly studied. The airway smooth muscle is an effector that controls the tracheal tube, and its pathological contraction is the ultimate factor in airway obstruction and dyspnea. It plays a key role in determining the effectiveness of asthma treatment. It is generally believed that the pathological contraction is an important pathological basis for the refractory asthma. The A disintegrin and metalloprotease33 (ADAM33) gene has been proved to be an asthma susceptible gene, and the expression level of the airway smooth muscle and basement membrane in most of the asthmatic patients is more than that of the healthy subjects. High, indicating that ADAM33 may be involved in the regulation of the mechanical environment of ASMC, which is related to the abnormal mechanical properties of ASMC and airway remodeling in asthmatic patients. However, there is no direct evidence to prove the relationship between the biomechanical behavior of ASMC and the expression of ADAM33. Therefore, the molecular biology method is used to regulate the ADAM33 expression of ASMC and to adopt a fine method. Cell biomechanical methods were used to measure the mechanical behavior of overexpression and silent ADAM33 ASMC, in order to reveal the direct relationship between the two. The main contents and results of this paper are as follows:
(1) the expression level of ADAM33 was significantly increased with the increase of the number of nebulization weeks in SD rats. First, using aluminum hydroxide as an adjuvant, ovalbumin (OVA) sensitized and stimulated asthma and established a model of asthma in SD (SpragueDawley) rats. OVA atomization 3 times a week, 12 weeks in a total of 12 weeks, and the lung function (P) was taken every 2 weeks. The determination of enh) and the observation of airway tissue HE (Hematoxylin-Eosin staining) staining showed that we had successfully constructed the rat model of asthma. Then, the quantitative RT-PCR and Western blot techniques were used to detect the expression of ADAM33 in the asthmatic smooth muscle cells of SD rats with different weeks of OVA atomization. The results showed that the ADAM33 expression level was with OVA atomization. The number of weeks increased significantly, and the quantitative RT-PCR was basically consistent with the experimental results of Western blot, indicating that the expression level of ADAM33 was positively correlated with the severity of asthma. This result was consistent with the research findings of Lee in the patients with asthma, and a closer step proved that the asthma model of the SD rat constructed in our experiment could be true. To simulate asthma.
(2) to obtain ADAM33 overexpression and silence ASMC. first, construct ADAM33 gene overexpression and interference with lentivirus recombinant vector, then transfection of lentivirus, with GFP empty and untransfected cells as control. The recombinant lentivirus transfected to normal SD rats ASMC, 24h after 24h, and GFP expression gradually increased, to GFP expression when 72h was cultured. When MOI (Multiplicity ofInfection) =100 was found, the efficiency of lentivirus infection ASMC was 95%. quantitative RT-PCR detection results. The expression of ADAM33 gene expression level in overexpression group was 8 times (P < 0.01), and pLVT453 silencing efficiency was up to 75% (P < 0.01) in silent group. The expression of ADAM33 protein showed that the expression of ADAM33 protein in the cells of the overexpression group was significantly higher than that in the two groups (P < 0.01), while the expression of the silent group decreased significantly (P < 0.01). The expression of ADAM33 protein in the two control groups was not significantly different. The stable ADAM33 overexpression and the silent ASMC were successfully obtained and the recombinant lentivirus was reorganized in this experiment. Transfection obviously increased or inhibited the expression of ADAM33 gene mRNA and protein in ASMC.
(3) the change of ADAM33 gene expression level changed the migration ability and proliferation ability of ASMC. The proliferation of different 4 groups was observed by MTT, and the proliferation ability of pLV-ADAM33 in the overexpression group was not significantly different from that in the blank control group, while the silent group pMa-ADAM33 was significantly lower than the cell proliferation ability of the blank group. The cell proliferation ability of the low (P < 0.05).GFP control group was significantly lower than that in the untransfected control group (P < 0.01). Therefore, we can see that the lentivirus transfection has a inhibitory effect on the cell proliferation ability. Although up regulation of ADAM33 has no difference in the cell proliferation ability to the untransfected control group, the cell proliferation ability of the GFP control group is significantly increased. Strong (P < 0.01). The results of detection of lateral migration of cells by scratch method (Wound healing) showed that there was no significant difference in the migration of 4 groups at 12h, but at 24h, the transversal migration of pLV-ADAM33 in the overexpression group was significantly increased (P < 0.01) (P < 0.05) in the two group (P < 0.05), while the cells in the silent group decreased significantly (P < 0.01). The results showed that the lateral migration of ASMC could be affected by specific changes in the expression of ADAM33 gene.
(4) ADAM33 gene can regulate the cellular mechanical properties of ASMC, such as cell base stiffness, contractile force and traction tension, etc. using optical capture (Optical trapping) and magnetic force torsional cell measurement (Magnetic TwistingCytometry) and Fu Liye transform traction microscopy (Fourier Transform Traction Microscopy, FTTM) detection by lentivirus transfection The biomechanical properties of the stable overexpression and interference of ADAM33 ASMC. The results showed: 1) the basal stiffness of pLV-ADAM33 in the ASMC group of ADAM33 overexpressed, the contractile force after the KCL agonist was significantly higher than that of the control group, while the above parameters in the ADAM33 silent cell group were significantly lower than that of the control.2) confocal immunofluorescence. The results show that there is a co localization relationship between ADAM33 and macula vinculin, and some ADAM33 has a distribution along the stress fiber, indicating that ADAM33 may regulate the cytoskeleton structure and the formation of.3 in the plaque. The changes in the expression level of the ADMA33 gene can regulate the cell mechanics related gene Calpon by the quantitative RT-PCR detection results. Expression of in and Integrin- beta 1.
From the above results, we speculate that the regulation of the expression of ADAM33 directly affects the biomechanical behavior of ASMCs, suggesting that the biomechanical behavior of ASMCs has a direct connection with the expression of ADAM33. Therefore, the abnormal expression of ADAM33 gene and the abnormality of the repair of the ADAM33 in the asthmatic patients may be played in the air channel remodeling and the hyperresponsiveness of the airway contraction. The decisive role is of profound significance in understanding the pathogenesis of asthma, especially the internal regulation mechanism of the airway function transformation, so as to explore effective targets and drugs for asthma treatment.

【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R562.25

【共引文献】