Bcl-2抑制剂诱导粒细胞凋亡缓解PM引起的气道炎症

发布时间：2018-04-29 13:28

  本文选题：PM + 气道炎症　； 参考：《浙江大学》2017年硕士论文

【摘要】：近年来,由于工业发展以及城市化进程的推进,环境污染越来越严重,越来越多的国内外研究表明,空气中一定浓度的颗粒物,对于一些呼吸系统疾病包括哮喘、COPD、肺癌以及心血管系统疾病的发病率的上升有着重要的影响。PM主要包括工业生产排放、自然源中土壤灰尘、机动车尾气或是生物燃料及矿物燃料燃烧所产生,PM颗粒物进入气道会通过堆积在气道或者通过支气管进入肺泡,从而能够引起许多呼吸系统及肺部疾病或者加重原有的气道炎症疾病。PM的危害与其颗粒物的大小有着必然的联系颗粒物的颗粒直径越小时,颗粒物的表面积就会越大而且其表面所附着的有毒物质也会增加,同时颗粒进入气道后的沉降速度也会变慢,同时进入肺泡的机会也会变大,对肺部的危害也会更严重。研究发现暴露于一定浓度颗粒物的环境时可诱导明显的气道高反应性,也会诱导以中性粒细胞为主的气道炎症;可加重哮喘气道炎症,从而导致哮喘患者对激素治疗敏感性降低,一定浓度颗粒物处理细胞也会增强气道上皮细胞内氧化应激水平从而诱导气道上皮间质的转化。目前有很多研究将诱导炎症细胞死亡而清除浸润的炎症细胞作为治疗炎症性疾病的一个重要的治疗方法。而且有研究表明凋亡抑制蛋白Bcl-2的抑制剂能够通过诱导嗜酸性粒细胞或者中性粒细胞的凋亡从而比较有效的治疗呼吸气道炎症例如哮喘特别是对于激素治疗抵抗的中性粒细胞型哮喘。本研究在浙江大学呼吸疾病研究所关于PM课题研究的基础上去探讨PM诱导的气道炎症是否与凋亡抑制蛋白Bcl-2有关系,并探讨凋亡抑制蛋白Bcl-2的抑制剂是否能够缓解PM诱导气道炎症从而开发治疗PM诱导气道炎症的新策略。目的:体内研究建立PM诱导气道炎症模型,研究凋亡抑制蛋白Bcl-2在PM诱导的气道炎症中的作用,并利用Bcl-2过表达小鼠去验证是否凋亡抑制蛋白在PM诱导的气道炎症中发挥重要作用同时利用Bcl-2抑制剂去处理体内PM诱导的气道炎症模型,探究其是否能够缓解此类炎症。方法:体外首先用标准化的PM气道滴注WT小鼠,建立气道炎症模型,流式细胞术运用AnnexiV/PI检测炎症细胞的凋亡,探究是否PM诱导的气道炎症与炎症细胞的凋亡有关。同时检测PM干预后小鼠肺泡灌洗液细胞中凋亡抑制蛋白Bcl-2的表达,探讨是否炎症细胞的凋亡与Bcl-2相关。然后利用Vav-Bcl-2转基因小鼠气道滴注PM,炎症Bcl-2在PM诱导的气道炎症中的作用。最后利用Bcl-2选择性抑制剂ABT199处理PM干预后的小鼠,看是否能够给缓解PM引起的气道炎症,并利用Annexi V/PI检测炎症细胞的凋亡从而确定ABT199是否是通过增加炎症细胞的凋亡从而缓解PM引起的气道炎症。结果:连续两天气道滴注PM会引起肺泡灌洗液总数、肺泡灌洗液中中性粒细胞比例明显增加,病理切片结果也也说明PM能够明显增加炎症细胞的聚集。Annexi V/PI检测炎症细胞的凋亡结果说明PM干预后炎症细胞的凋亡与NS组相比明显增加。且IF及流式检测结果也表明BALF细胞中凋亡抑制蛋白Bcl-2表达也有明显增加。Bcl-2过表达小鼠在PM气道滴注后与WT小鼠相比,中性粒细胞数目、气道炎症的聚集及中性粒细胞趋化因子的表达都明显增加而且炎症自行减退的时间及程度都减慢。构建模型时在末次滴注PM之后24小时滴注Bcl-2选择性抑制剂,结果发现Bcl-2抑制剂能够明显诱导中性粒细胞的凋亡从而减轻PM引起的气道炎症。结论:PM能够诱导以中性粒细胞为主的气道炎症,并且这种气道炎症在Bcl-2过表达的小鼠体内会明显加重且自行清理的时间延长,Bcl-2选择性抑制剂ABT-199能够通过诱导中性粒细胞的凋亡即炎症细胞的凋亡从而减轻PM引起的气道炎症。
[Abstract]:In recent years, due to the development of industry and the advancement of urbanization, environmental pollution is becoming more and more serious. More and more domestic and foreign studies have shown that certain concentrations of particulate matter in the air have an important impact on the rise of some respiratory diseases including asthma, COPD, lung cancer and cardiovascular disease..PM mainly includes work. Production of industrial emissions, natural sources of soil dust, motor vehicle exhaust, or combustion of biofuels and fossil fuels, and PM particles entering the airway by accumulating in the airway or through the bronchus into the alveoli, which can cause many respiratory and pulmonary diseases or the damage to the original airway inflammation,.PM, and its grain. The larger the particle size is, the larger the particle size, the larger the surface area of the particles and the increase of the toxic substances attached to the surface, and the sedimentation rate will slow down after the particles enter the airway, and the chances of entering the alveoli will also become larger, and the damage to the lungs will be more serious. When exposed to a certain concentration of particulate matter, it can induce obvious airway hyperresponsiveness, and also induce airway inflammation dominated by neutrophils, which can aggravate airway inflammation in asthmatic patients, resulting in a decrease in sensitivity to hormone therapy in asthmatic patients. Certain concentration of granular substance treatment cells also enhance the level of oxidative stress within the airway epithelial cells. There are many studies that will induce the death of inflammatory cells and remove infiltrating inflammatory cells as an important treatment for the treatment of inflammatory diseases. Moreover, studies have shown that inhibitors of apoptosis inhibitory protein Bcl-2 can induce apoptosis of eosinophils or neutrophils by inducing apoptosis from eosinophilic granulocytes or neutrophils It is more effective to treat respiratory airway inflammation, such as asthma, especially for neutrophilic granulocytic asthma, which is resistant to hormone therapy. This study is based on the study of PM in the respiratory disease Research Institute of Zhejiang University to investigate whether PM induced airway inflammation is associated with apoptosis suppressor Bcl-2 and to explore the apoptosis suppressor protein Bcl-2 Whether or not the inhibitors can relieve the PM induced airway inflammation and develop a new strategy for the treatment of airway inflammation by PM. Objective: to establish a PM induced airway inflammation model in vivo, to study the role of apoptosis inhibitory protein Bcl-2 in the airway inflammation induced by PM, and to verify whether the apoptosis inhibitory protein is induced by PM in the PM induced by the Bcl-2 overexpressed rat. The airway inflammation plays an important role and the Bcl-2 inhibitor is used to deal with the PM induced airway inflammation model in vivo, and to explore whether it can relieve this kind of inflammation. Methods: in vitro, WT mice were injected with standardized PM airway in vitro to establish the airway inflammation model. Flow cytometry was used to detect the apoptosis of inflammatory cells by AnnexiV/PI, and to explore if PM was used. The induced airway inflammation is related to the apoptosis of inflammatory cells. At the same time, the expression of apoptosis suppressor protein Bcl-2 in the alveolar lavage cells of mice after PM was detected, and the apoptosis of the inflammatory cells was related to the Bcl-2. Then, PM was injected into the airway of the Vav-Bcl-2 transgenic mice, and the effect of inflammatory Bcl-2 in the airway inflammation induced by PM was used. Finally, the effect of PM on the airway inflammation induced by PM was used. Bcl-2 selective inhibitor ABT199 treatment of PM dry mice, see whether it can relieve airway inflammation caused by PM, and use Annexi V/PI to detect the apoptosis of inflammatory cells to determine whether ABT199 is by increasing the apoptosis of inflammatory cells to alleviate airway inflammation caused by PM. Fruit: two days of continuous infusion of PM in the airway will cause alveolar irrigation. The total number of lotion and the proportion of neutrophils in the alveolar lavage fluid increased obviously. The pathological section also showed that PM could significantly increase the aggregation of inflammatory cells.Annexi V/PI to detect the apoptotic results of inflammatory cells, indicating that the apoptosis of inflammatory cells increased significantly after PM intervention. And IF and flow detection results also indicated that BALF cells were withered in BALF cells. The expression of Bcl-2 also significantly increased the number of neutrophils, the aggregation of neutrophils and the expression of neutrophil chemotactic factor in the.Bcl-2 overexpressed mice compared with the WT mice, and the time and degree of inflammation self-reduction slowed down. 24 hours after the final infusion of PM, 24 hours after the final infusion of the model was constructed. Bcl-2 selective inhibitors were instilled. The results showed that Bcl-2 inhibitors could induce apoptosis of neutrophils and reduce airway inflammation caused by PM. Conclusion: PM can induce airway inflammation based on neutrophil, and this airway inflammation can be significantly increased in Bcl-2 overexpressed mice and the time of self-cleaning is prolonged, Bc ABT-199, a selective inhibitor of L-2, can alleviate airway inflammation caused by PM by inducing apoptosis of neutrophils, namely apoptosis of inflammatory cells.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R56
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本文编号：1820124