骨化三醇调节慢性哮喘小鼠气道重塑的机制研究

发布时间：2018-10-26 18:23

【摘要】：近年来，，作为哮喘慢性化、严重化、持续化的病理基础，气道重塑在哮喘防治中的潜在作用被广为关注。它不仅是激素抵抗性哮喘的病理基础，也是不可逆性气道阻塞和气道高反应性的病理基础。作为Vit-D在体内极其重要的的代谢产物之一，骨化三醇（罗盖全，Rocalirol）在哮喘的治疗中不仅对于气道慢性炎症而且对于免疫应答均起到一定的调节作用。但至今国内外均未见关于骨化三醇对哮喘气道重塑的报道。该研究设计从体内、外两个方面观察骨化三醇对支气管哮喘气道重塑的影响，并进一步探讨其作用机制，为推广其临床应用提供依据。该项内容分为两个部分：第一部分骨化三醇调节哮喘小鼠气道重塑及NF-κB信号通路的研究目的：观察骨化三醇对哮喘小鼠气道重塑的影响，并进一步探讨其机制。方法：实验小鼠随机分为对照组、哮喘组和VD组。采用卵白蛋白致敏及激发的方法制备小鼠哮喘气道重塑的模型；选择HE染色法观察气道壁病理改变；Masson染色观察气道壁胶原染色情况，AB-PAS染色观察杯状细胞增生情况，计算机图像分析系统测定气道形态学指标，蛋白免疫印记法检测NF-κB p65的蛋白水平。结果：(1)骨化三醇对哮喘气道重塑具有部分逆转的作用；(2)骨化三醇能明显抑制哮喘肺组织NF-κB p65的核易位。结论：骨化三醇的干预可显著减轻哮喘气道重塑的病理改变；这一作用可能与其对哮喘肺内NF-κB活化的抑制作用有关。第二部分骨化三醇对哮喘小鼠气道平滑肌细胞凋亡及NF-κB信号通路的影响目的：检测骨化三醇对哮喘小鼠气道平滑肌细胞（airway smooth musclecells,ASMCs）的凋亡及其核转录因子-κB信号通路的影响，从细胞及分子两个水平探究Rocalirol对哮喘气道重塑的可能作用机制。方法：原代培养正常及哮喘小鼠ASMCs，以Rocalirol作为干预因素。流式细胞仪测定细胞凋亡，荧光显微镜观察细胞凋亡形态学变化；凝胶迁移滞后实验(EMSA法)检测NF-κB的DNA结合活性；实时荧光定量RT-PCR法和蛋白免疫印记法检测IκBα的表达。结果：(1)10 7M Rocalirol可显著诱导哮喘小鼠ASMCs的凋亡；(2) Rocalirol可显著削弱哮喘小鼠ASMCs中NF-κB的DNA结合活性；(3)Rocalirol可增加哮喘小鼠ASMCs中IκBα的蛋白及mRNA表达。结论：Rocalirol可直接诱导哮喘ASMCs的凋亡，这种作用可能与NF-κB信号通路受骨化三醇抑制有关。
[Abstract]:In recent years, as the pathological basis of chronic, severe and persistent asthma, the potential role of airway remodeling in the prevention and treatment of asthma has been widely concerned. It is not only the pathological basis of hormone resistant asthma, but also the pathological basis of irreversible airway obstruction and airway hyperresponsiveness. As one of the most important metabolites of Vit-D in vivo, roguetriol (, Rocalirol) plays an important role not only in chronic airway inflammation but also in immune response in the treatment of asthma. However, there is no report on airway remodeling of asthma by ossifying triol at home and abroad. The purpose of this study was to investigate the effect of ossifying triol on airway remodeling of bronchial asthma in vivo and in vitro, and to explore its mechanism. The content is divided into two parts: the first part is the effect of ossifying triol on airway remodeling and NF- 魏 B signaling pathway in asthmatic mice objective: to observe the effect of ossifying triol on airway remodeling in asthmatic mice. The mechanism is further discussed. Methods: the mice were randomly divided into control group, asthma group and VD group. The airway remodeling model of mouse asthma was established by sensitizing and stimulating ovalbumin, and the pathological changes of airway wall were observed by HE staining. Collagen staining in airway wall was observed by Masson staining, goblet cell proliferation was observed by AB-PAS staining, airway morphology was measured by computer image analysis system, and the protein level of NF- 魏 B p65 was detected by protein imprinting. Results: (1) ossifying triol could partially reverse airway remodeling of asthma, (2) ossification triol could significantly inhibit the nuclear translocation of NF- 魏 B p65 in lung tissue of asthma. Conclusion: the intervention of calcitriol can significantly attenuate the pathological changes of airway remodeling in asthma, which may be related to its inhibitory effect on the activation of NF- 魏 B in the lung of asthma. Part two effects of ossifying triol on apoptosis of airway smooth muscle cells and NF- 魏 B signaling pathway in asthmatic mice objective: to detect the effect of oscification triol on (airway smooth musclecells, of airway smooth muscle cells of asthmatic mice ASMCs) apoptosis and the effect of nuclear transcription factor-魏 B signaling pathway. To explore the possible mechanism of Rocalirol on airway remodeling in asthmatic patients at both cellular and molecular levels. Methods: Rocalirol was used as an intervention factor in primary cultured normal and asthmatic mice ASMCs,. Apoptosis was measured by flow cytometry, morphological changes of apoptosis were observed by fluorescence microscope, DNA binding activity of NF- 魏 B was detected by gel migration lag assay (EMSA). The expression of I 魏 B 伪 was detected by real time fluorescence quantitative RT-PCR and protein imprinting. Results: (1) 10 渭 m Rocalirol significantly induced the apoptosis of ASMCs in asthmatic mice; (2) Rocalirol significantly reduced the DNA binding activity of NF- 魏 B in ASMCs of asthmatic mice; (3) Rocalirol increased the expression of I 魏 B 伪 and mRNA in ASMCs of asthmatic mice. Conclusion: Rocalirol can directly induce the apoptosis of ASMCs in asthma. This effect may be related to the inhibition of NF- 魏 B signaling pathway by oscitic triol.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R562.25

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