模拟病理状态下气道上皮组织对气道平滑肌增生的影响研究

发布时间：2018-10-20 08:18

【摘要】：哮喘（Asthma）作为一种慢性呼吸道疾病，正日益成为严重威胁人类健康的重大问题。令人遗憾的是，科学界对哮喘病的病因和发展过程至今还没有完全认识清楚，其病理机制也一直是医学上的重要难题。气道是气体进出肺的通道。有哮喘的人呼吸道发炎，这使得呼吸道变肿胀也非常敏感。他们往往对某些吸入物质反应强烈。当哮喘发生时，气道周围的肌肉收紧，气道缩小，造成流入肺部的气体不足，甚至肿胀会进一步恶化，使气道更窄。气道平滑肌的增生则是哮喘的主要发病症状之一，有报道证明在哮喘个体中肌细胞的增生速度远高于非哮喘个体。虽然皮质类固醇通过减少细胞周期蛋白D1的表达以及视网膜母细胞瘤蛋白磷酸化，能抑制正常气道中肌细胞的增生，但是在哮喘个体中却没有效果。尽管做了如此多的研究，但是关于气道平滑肌增生还有很多问题额待解决。气道上皮组织在呼吸道系统的防御中起着至关重要的作用，气道上皮组织通过其拦截系统阻止外源有害刺激对气道上皮的损害，从而保护呼吸道，上皮脱落(特别是在痰中有成团的上皮细胞时)，以及支气管中的上皮脱落是上皮受损的明显现象，在接下来的上皮修复过程中，气道上皮会分泌促进细胞生长、迁移的信号因子，这些因子中很多都能刺激气道平滑肌的增生。气道中无论是气道平滑肌还是气道上皮在哮喘发生时都有显著的病理特征，但是两者之间存在怎样的信号交流，目前还没有完整的理论解释。目前研究人员做了大量关于气道平滑肌的研究，但是关于气道上皮组织对气道平滑肌增生的影响尚未有报道。本课题组在体外用多聚左旋精氨酸(poly—L—arginine，PLA)构建气道上皮细胞（AEC）损伤模型，并与正常平滑肌细胞(ASMC)共培养（在哮喘发病过程中，嗜酸性粒细胞是主要的效应细胞，嗜酸性粒细胞阳离子蛋白(ECP)是其主要效应成分。PLA作为ECP的类似物，可以在体外模拟细胞受损伤的状态）。而为了更真实地模拟哮喘发生时气道上皮组织的状况，本课题还在共培养过程中对AEC施加外部压力。最后检测共培养系统中ASMC增殖情况，从而探索病理状态下气道上皮组织对气道平滑肌增生的影响作一个初步探索： ①为了构建体外模型：本课题在体外收集并培养AEC和ASMC，并用形态学和免疫荧光学方法对收集到的细胞进行鉴定。鉴定结果表明，用组织块贴壁法获得了大量的原代ASMC，用胰蛋白酶冷消化法获得了大量的AEC。 ②PLA对大鼠气道上皮细胞活性影响研究：设置0-50μM（每5μM浓度差为一个间隔梯度）共10个浓度梯度的PLA对AEC进行处理，处理时间为24h。然后用台盼蓝进行细胞计数。最后结果显示，与对照组比较，即使小浓度的PLA（5μM）对气道上皮细胞也有明显的毒性作用（P0.01）.而在PLA浓度大于20μM时，毒性作用的变化不再明显（PLA浓度20μM时各组分之间的P0.05）. ③AEC损伤模型构建：对AEC用高浓度（50μM）PLA进行短时间处理（10分钟），构建气道上皮细胞损伤模型。 ④共培养装置的设计：为了在共培养时能对气道上皮细胞进行单独施压，我们改进了共培养的压力装置。最后，我们在设计的装置能提供我们需要的稳定压力（14KPa）。 ⑤本实验设置5个实验组： ASMC单独培养为对照组（A）、ASMC与AEC共培养（B）、ASMC与受损伤的AEC共培养（C）、 ASMC与未受损伤的AEC共培养并施加外部压力(D)、ASMC与受损伤的AEC共培养并施加外部压力(E) ⑥共培养条件下ASMC增值检测：每12h用MTT测量一次ASMC的生长OD值，共取5个时间点，既12h、24h、36h、48h、60h分别测量一次每组细胞的OD值，用软件进行相关的分析结果显示：60h时，BCDE组与A组以及BCDE组之间p均0.01.E组的OD最大， E组中ASMC的生长速度明显高于其他组。而E组与D组以及E组与C组比较都有显著性差异（p0.01）,说明了外部压力和AEC损伤对ASMC的增值有独立的作用。我们最后得出结论，在体外研究中，气道上皮在损伤和接受外部压力刺激后，加剧了气道平滑肌的增生。
[Abstract]:Asthma (Astma), as a chronic respiratory disease, is becoming a serious threat to human health. Unfortunately, the scientific community has not yet fully recognized the etiology and development of asthma, and its pathological mechanism has always been an important issue in medicine. The airway is gas in and out of the lungs Channel: Inflammation of the respiratory tract of a person with asthma, which causes the respiratory tract to become swollen and swollen. Sensitive. They tend to react to certain inhaled substances Severe. When asthma occurs, the muscles around the airway tighten, the airway is deflated, causing insufficient gas flowing into the lungs, and even swelling will further deteriorate, causing the airway More narrow. The proliferation of airway smooth muscle is one of the main symptoms of asthma. It is reported that myocyte proliferation is much higher in asthmatic individuals. Non-asthmatic individuals. Although corticosteroids inhibit myocyte proliferation in normal airways by reducing the expression of cyclin D1 and phosphorylation of retinoblastoma proteins, in asthma individuals No effect. Despite so many studies, there are many questions about airway smooth muscle proliferation The upper airway of the airway plays a vital role in the defense of the respiratory tract system, which prevents the damage to the airway epithelium through its intercepting system, thus protecting the respiratory tract and the epithelium (especially in the sputum). When the epithelial cells of the group), and the epithelial exfoliation in the bronchi are the obvious phenomena of epithelial damage, in the next epithelial tissues, the airway epithelium will secrete signaling factors that promote cell growth and migration, and many of these factors can stimulate the epithelial cells. Airway smooth muscle hyperplasia, airway smooth muscle or airway epithelium have significant pathological characteristics in the occurrence of asthma, but there is signal communication between them. There is no complete theoretical explanation. Now researchers have done a lot of research on airway smooth muscle, but on the airway smooth muscle is smooth In vitro, we constructed airway epithelial cell (AEC) injury model with poly L arginine (PLA) in vitro and co-cultured with normal smooth muscle cells (ASMC) (acidophilic in the course of asthma). Grangranulocytes are the main effector cells, eosinophil cationic proteins (E CP) is its main effect component. As an ECP analog, PLA can be used in vitro The subject is also co-cultured in order to simulate the condition of the airway epithelium in the event of asthma more realistically. In order to explore the proliferation of ASMC in co-culture system, the proliferation of ASMC in co-culture system was detected. In order to build an in vitro model: this topic is to build an in vitro model: AEC and ASMC were collected and cultured, and morphology and immunofluorescence were used The results showed that a large amount of primary ASMC was obtained by tissue block attachment, and pancreatic protein was used. A large number of AEC was obtained by the enzyme-cold digestion method. The effects of PLA on the activity of airway epithelial cells in rats were studied: a total of 10 concentrations of PLA with a concentration of 0-50. m u.M (one interval gradient per 5. mu.M concentration) were set. Processing AEC, processing time Cell counts were then performed with trypan blue. The results showed that, compared with the control group, even small concentrations of PLA (5. mu.M) were fine for the airway epithelium. There were also significant toxic effects on the cells (P0.01). However, when the concentration of PLA was greater than 20. m u.M, the change of toxicity was no longer significant (PLA concentration). P0. 05) between the components at 20 & mu; M. AEC damage model construction: AEC high concentration (50. mu.M) PLA for a short time Histology (10 minutes) to construct an airway epithelial cell damage model. Design of a co-culture device: To be able to treat the airway during co-culture The skin cells were pressed separately, and we improved the pressure device for co-culture. Finally, we're setting up The device provided by the invention can provide the stable pressure required by us (14KPa), and five experimental groups are arranged in the experiment: the ASMC is independently cultured as a control group (A), the ASMC is co-cultured with the AEC (B), the ASMC is co-cultured with the damaged AEC (C), ASMC co-culture with undamaged AEC and external pressure applied (D) ASMC was co-cultured with the damaged AEC and applied external pressure (E) under co-culture conditions for ASMC value-added detection: the growth OD value of an ASMC was measured by MTT every 12h, and 5 time points were taken, not only 12h, The OD values of each group of cells were measured at 24h, 36h, 48h and 60h, respectively. The correlation analysis results showed that when 60h, the p between the group A and the group A and the group B was 0. 0. 1. The OD of group E was the largest, and the growth rate of ASMC in group E was significantly higher than that in other groups. It is shown that external pressure and AEC damage have an independent effect on the value-added value of ASMC. We conclude that in vitro research
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25

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