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  本文關(guān)鍵詞：布地奈德對哮喘小鼠氣道上皮occludin和E-cadherin表達的影響　出處：《瀘州醫(yī)學(xué)院》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 哮喘 氣道上皮 閉鎖蛋白 E-鈣粘蛋白 布地奈德

【摘要】：目的：觀察哮喘小鼠氣道上皮閉鎖蛋白(occludin)和E-鈣粘蛋白(E-cadherin)的表達和布地奈德(BUD)對其表達的影響，探討occludin和E-cadherin在哮喘發(fā)病中的作用及糖皮質(zhì)激素治療哮喘的機制。方法：將24只SPF級雌性BALB/c小鼠隨機分成哮喘組、布地奈德治療組(BUD組)及對照組，每組8只。哮喘組：0、7、14d腹腔注射新鮮配制的卵蛋白(OVA)+氫氧化鋁的生理鹽水(NS)混懸溶液0.2ml [含OVA20μg,氫氧化鋁2mg]致敏,第21d起給予1%OVA霧化吸入激發(fā)30min,1次/d,連續(xù)14d，每次激發(fā)前1h霧化吸入NS2ml。BUD組：致敏及激發(fā)同哮喘組，但每次激發(fā)前1h霧化吸入布地奈德混懸液1mg(2ml)。對照組：用相同的方法以NS代替OVA進行致敏和激發(fā)。小鼠末次激發(fā)24h后處死，蘇木精-伊紅(HE)染色觀察支氣管肺組織病理學(xué)的改變；電鏡觀察氣道上皮的超微結(jié)構(gòu)；過碘酸-雪夫氏染色(PAS)法觀察氣道黏膜上皮層杯狀細胞增生和黏液分泌情況；支氣管肺泡灌洗液(BALF)行嗜酸性粒細胞計數(shù)；免疫組織化學(xué)染色法觀察氣道上皮occludin和E-cadherin的表達；實時熒光定量PCR(Real-timePCR)檢測occludin mRNA的表達。結(jié)果：⑴HE染色：哮喘組小鼠氣道上皮細胞壞死脫落，支氣管黏膜層大量炎癥細胞浸潤，，黏液腺增生，基底膜增厚，氣道管腔狹窄，部分管腔內(nèi)可見黏液栓；BUD組上述病理改變較哮喘組減輕；對照組未見上述病理改變。3組小鼠的Underwood肺組織病理學(xué)評分分別為哮喘組(4.1±0.675)、BUD組(2.3±0.736)和對照組(0.5±0.506)，三組比較差異具有統(tǒng)計學(xué)意義(P0.05)，BUD組評分比哮喘組低（P0.05），但高于對照組(P0.05)。⑵電鏡：哮喘組氣道上皮細胞纖毛明顯脫落、變稀、變短，微絨毛減少。上皮細胞間的連接結(jié)構(gòu)有松裂或缺失，細胞間隙增寬，基底膜不連續(xù)，膠原纖維增多。BUD組上述表現(xiàn)較哮喘組有所減輕。對照組未見上述改變。⑶PAS染色：與對照組(0.250±0.463)比較，哮喘組氣道上皮PAS染色陽性的杯狀細胞數(shù)(3.375±0.744)明顯增多，差異有統(tǒng)計學(xué)意義(P0.01)；與哮喘組比較，BUD組杯狀細胞數(shù)(1.625±0.518)增生減少，但仍高于對照組，差異有統(tǒng)計學(xué)意義(P0.01)。⑷BALF中嗜酸性粒細胞（EOS）計數(shù)：與對照組(0.750±0.707)比較，哮喘組EOS數(shù)量(20.875±1.808)明顯增加，差異具有統(tǒng)計學(xué)意義(P0.01)；與哮喘組比較，BUD組EOS數(shù)量(10.625±1.302)減少，但仍高于對照組，差異有統(tǒng)計學(xué)意義(P0.01)。⑸免疫組織化學(xué)染色：對照組小鼠occludin、E-cadherin主要在氣道上皮細胞表面和細胞與細胞間的包膜上呈連續(xù)的強陽性表達，而BUD組表達強度減弱，為不連續(xù)表達，但仍比哮喘組表達明顯。哮喘組occludin的平均光密度值(OD值)(0.126±0.032)明顯低于對照組(0.652±0.313)及BUD組(0.337±0.028)，差異具有統(tǒng)計學(xué)意義(P0.01)；與對照組比較，BUD組OD值仍減小，差異有統(tǒng)計學(xué)意義(P0.01)。哮喘組E-cadherin的OD值(0.173±0.043)低于對照組(0.656±0.068)及BUD組(0.288±0.049)，差異具有統(tǒng)計學(xué)意義(P0.01)，BUD組的OD值仍較對照組低，差異具有統(tǒng)計學(xué)意義(P0.01)。⑹Real-time PCR：三組小鼠支氣管肺組織的occludin mRNA相對含量分別為(7.200±4.959)、(3.127±2.030)、(4.772±1.848)，三組之間比較差異無統(tǒng)計學(xué)意義(F=1.960，P0.05)。結(jié)論：⑴成功建立了BALB/c小鼠哮喘模型；⑵哮喘小鼠存在氣道炎癥和氣道上皮連接結(jié)構(gòu)的損傷；⑶哮喘小鼠氣道上皮occludin和E-cadherin的表達降低，可能參與了哮喘的發(fā)病過程；⑷布地奈德能上調(diào)哮喘小鼠氣道上皮occludin和E-cadherin的表達，對氣道上皮屏障結(jié)構(gòu)具有修復(fù)作用，可能是糖皮質(zhì)激素治療哮喘的機制之一。
[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.

【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R562.25

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