布地奈德对哮喘小鼠气道上皮occludin和E-cadherin表达的影响

发布时间：2018-01-14 01:13

本文关键词：布地奈德对哮喘小鼠气道上皮occludin和E-cadherin表达的影响　出处：《泸州医学院》2014年硕士论文　论文类型：学位论文

【摘要】：目的：观察哮喘小鼠气道上皮闭锁蛋白(occludin)和E-钙粘蛋白(E-cadherin)的表达和布地奈德(BUD)对其表达的影响，探讨occludin和E-cadherin在哮喘发病中的作用及糖皮质激素治疗哮喘的机制。方法：将24只SPF级雌性BALB/c小鼠随机分成哮喘组、布地奈德治疗组(BUD组)及对照组，每组8只。哮喘组：0、7、14d腹腔注射新鲜配制的卵蛋白(OVA)+氢氧化铝的生理盐水(NS)混悬溶液0.2ml [含OVA20μg,氢氧化铝2mg]致敏,第21d起给予1%OVA雾化吸入激发30min,1次/d,连续14d，每次激发前1h雾化吸入NS2ml。BUD组：致敏及激发同哮喘组，但每次激发前1h雾化吸入布地奈德混悬液1mg(2ml)。对照组：用相同的方法以NS代替OVA进行致敏和激发。小鼠末次激发24h后处死，苏木精-伊红(HE)染色观察支气管肺组织病理学的改变；电镜观察气道上皮的超微结构；过碘酸-雪夫氏染色(PAS)法观察气道黏膜上皮层杯状细胞增生和黏液分泌情况；支气管肺泡灌洗液(BALF)行嗜酸性粒细胞计数；免疫组织化学染色法观察气道上皮occludin和E-cadherin的表达；实时荧光定量PCR(Real-timePCR)检测occludin mRNA的表达。结果：⑴HE染色：哮喘组小鼠气道上皮细胞坏死脱落，支气管黏膜层大量炎症细胞浸润，，黏液腺增生，基底膜增厚，气道管腔狭窄，部分管腔内可见黏液栓；BUD组上述病理改变较哮喘组减轻；对照组未见上述病理改变。3组小鼠的Underwood肺组织病理学评分分别为哮喘组(4.1±0.675)、BUD组(2.3±0.736)和对照组(0.5±0.506)，三组比较差异具有统计学意义(P0.05)，BUD组评分比哮喘组低（P0.05），但高于对照组(P0.05)。⑵电镜：哮喘组气道上皮细胞纤毛明显脱落、变稀、变短，微绒毛减少。上皮细胞间的连接结构有松裂或缺失，细胞间隙增宽，基底膜不连续，胶原纤维增多。BUD组上述表现较哮喘组有所减轻。对照组未见上述改变。⑶PAS染色：与对照组(0.250±0.463)比较，哮喘组气道上皮PAS染色阳性的杯状细胞数(3.375±0.744)明显增多，差异有统计学意义(P0.01)；与哮喘组比较，BUD组杯状细胞数(1.625±0.518)增生减少，但仍高于对照组，差异有统计学意义(P0.01)。⑷BALF中嗜酸性粒细胞（EOS）计数：与对照组(0.750±0.707)比较，哮喘组EOS数量(20.875±1.808)明显增加，差异具有统计学意义(P0.01)；与哮喘组比较，BUD组EOS数量(10.625±1.302)减少，但仍高于对照组，差异有统计学意义(P0.01)。⑸免疫组织化学染色：对照组小鼠occludin、E-cadherin主要在气道上皮细胞表面和细胞与细胞间的包膜上呈连续的强阳性表达，而BUD组表达强度减弱，为不连续表达，但仍比哮喘组表达明显。哮喘组occludin的平均光密度值(OD值)(0.126±0.032)明显低于对照组(0.652±0.313)及BUD组(0.337±0.028)，差异具有统计学意义(P0.01)；与对照组比较，BUD组OD值仍减小，差异有统计学意义(P0.01)。哮喘组E-cadherin的OD值(0.173±0.043)低于对照组(0.656±0.068)及BUD组(0.288±0.049)，差异具有统计学意义(P0.01)，BUD组的OD值仍较对照组低，差异具有统计学意义(P0.01)。⑹Real-time PCR：三组小鼠支气管肺组织的occludin mRNA相对含量分别为(7.200±4.959)、(3.127±2.030)、(4.772±1.848)，三组之间比较差异无统计学意义(F=1.960，P0.05)。结论：⑴成功建立了BALB/c小鼠哮喘模型；⑵哮喘小鼠存在气道炎症和气道上皮连接结构的损伤；⑶哮喘小鼠气道上皮occludin和E-cadherin的表达降低，可能参与了哮喘的发病过程；⑷布地奈德能上调哮喘小鼠气道上皮occludin和E-cadherin的表达，对气道上皮屏障结构具有修复作用，可能是糖皮质激素治疗哮喘的机制之一。
[Abstract]:Objective: To observe the effect of airway epithelial occludin (occludin) and E- cadherin (E-cadherin) expression and effect of budesonide (BUD) on the expression and mechanism of occludin and E-cadherin in the pathogenesis of asthma and the role of glucocorticoids in the treatment of asthma. Methods: 24 SPF female BALB/c mice were randomly divided into asthma group budesonide, treatment group (BUD group) and control group, 8 rats in each group. The asthma group: 0,7,14d intraperitoneal injection of freshly prepared ovalbumin (OVA) saline + aluminum hydroxide (NS) suspension solution containing OVA20 0.2ml [g, aluminum hydroxide 2mg] sensitized 21d, given aerosol inhalation of 1%OVA 30min. 1 /d, continuous 14d, before each excitation 1H inhalation group NS2ml.BUD sensitized and challenged with asthma group, but before each excitation 1H inhalation of budesonide suspension (1mg 2ml). The control group: use the same method to replace the OVA sensitized NS And excited. The mice were sacrificed 24h after the last challenge, hematoxylin eosin (HE) staining of lung tissue pathological changes; ultrastructure of airway epithelial electron microscopy; periodic acid Schiff's stain (PAS) method was used to observe the airway mucosal epithelium goblet cell hyperplasia and mucus secretion; bronchoalveolar lavage fluid (BALF) for eosinophil count; immunohistochemical staining was used to observe the expression of occludin in airway epithelia and E-cadherin; real-time fluorescence quantitative PCR (Real-timePCR) to detect the expression of occludin mRNA. Results: the HE staining: the airway epithelial cells of asthmatic mice, necrosis of bronchial mucosa layer shedding, inflammatory cell infiltration, mucus gland hyperplasia, basement membrane thickening, airway stenosis, partial lumen in the mucus; in BUD group the pathological changes alleviated; the control group had no pathological changes in.3 group Und The lung tissue pathology erwood scores were asthma group (4.1 + 0.675), BUD group (2.3 + 0.736) and control group (0.5 + 0.506), with significant differences between the three groups (P0.05), BUD group score than asthma group (P0.05), but lower than that of the control group (P0.05) and electron microscopy. The cilia group: epithelial cells of asthmatic airway was falling, thinning, shorter, reduced microvilli. Connections between epithelial cells structure broken or missing, cell gap widened, the basement membrane is continuous, collagen fibers increased in.BUD group compared with the asthma group, the performance has been reduced. The control group had no change. PAS staining: and the control group (0.250 + 0.463), PAS group of asthmatic airway epithelial goblet cells staining positive number (3.375 + 0.744) increased significantly, the difference was statistically significant (P0.01); compared with the asthma group, BUD group, the number of goblet cells (1.625 + 0.518) proliferation decreased, but still higher than the control group, the difference is statistics Meaning (P0.01). The BALF in eosinophil (EOS) count: compared with the control group (0.750 + 0.707), the number of asthma group EOS (20.875 + 1.808) was significantly increased, the difference was statistically significant (P0.01); compared with the asthma group, the number of BUD group EOS (10.625 + 1.302) less. But still higher than the control group, the difference was statistically significant (P0.01). The immunohistochemical staining: control group mice occludin, E-cadherin showed strong positive expression in continuous airway epithelial cell surface and between cell and cell envelope, and group BUD expression intensity for discontinuous expression, but still higher than the asthma group the expression is obvious. The average optical density value of occludin in asthma group (OD) (0.126 + 0.032) was significantly lower than the control group (0.652 + 0.313) and BUD group (0.337 + 0.028), the difference was statistically significant (P0.01); compared with the control group, the OD value of BUD group is decreased, the difference was statistically significant (P0.01). Asthma The OD value of E-cadherin asthmatic group (0.173 + 0.043) is lower than that of the control group (0.656 + 0.068) and BUD group (0.288 + 0.049), the difference was statistically significant (P0.01), BUD group was still lower than the control group, the difference was statistically significant (P0.01). 6 Real-time PCR: the relative content of the three groups of mice in bronchiai occludin in the lung tissue of mRNA were (7.200 + 4.959), (3.127 + 2.030), (4.772 + 1.848), the difference between the three groups was not statistically significant (F=1.960, P0.05). Conclusion: BALB/c mouse asthma model was successfully established; the existence of asthma mice airway inflammation and airway epithelial junction structure damage; the decreased expression of occludin and E-cadherin in asthmatic mice airway epithelium, may be involved in the pathogenesis of asthma; the budesonide could up regulate the expression of occludin and E-cadherin in asthmatic mice airway epithelium, with repair of airway epithelial barrier structure, may be sugar Corticosteroids are one of the mechanisms for the treatment of asthma.

【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R562.25

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