【作者】 刘维克；

【导师】 徐青；

【作者基本信息】 河北医科大学 ， 儿科学， 2010， 硕士

【摘要】 目的:支气管哮喘(bronchial astham)是世界医学界公认的四大顽症之一,被列为十大死亡原因之最。目前,随着人们生活环境的改变,发病率逐年上升。其临床表现为突然而反复发作的喘息,呼吸困难,胸闷和咳嗽。气道炎症与气道重塑是支气管哮喘发病的病理基础。本实验通过建立哮喘模型,观察哮喘大鼠吸入布地奈德后血清中LI-25与IL-12比值的变化,探讨哮喘时免疫失衡与NF-κB表达的动态关系。观察哮喘大鼠吸入布地奈德后气道NF-κB,MMP-9表达,探讨哮喘时气道重塑与NF-κB的关系。方法:1动物与药品Wistar雄性大鼠30只,由河北省实验动物中心提供(实验动物质量合格证编号:903010);布地奈德由阿斯利康制药公司生产;免疫组化试剂盒,一抗cox2兔抗IgG多克隆抗体、购于boshide公司,SP9001试剂盒及DAB显色剂购于北京中衫生物技术有限公司。酶联免疫试剂盒购于美国RB公司;其他实验药品由河北医科大学免疫教研室提供。2模型制备及实验分组用随机数字法将30只雄Wistar大鼠随机分为3组:正常对照组、哮喘模型组、布地奈德治疗组,每组10只。哮喘大鼠模型的制备分致敏和激发两步。除正常对照组外,其余2组于第1天、第8天、第15天分3次腹腔注射抗原液1ml(含卵蛋白100mg、氢氧化铝干粉100mg)致敏,正常对照组用等量的生理盐水进行致敏;第21天开始将大鼠放入自制的不完全封闭的有机玻璃箱内,哮喘模型组和布地奈德治疗组予1%卵蛋白(OVA)溶液用空气压缩泵喷雾激发,每天1次,每次20分钟,连续激发7天,布地奈德治疗组于激发后,均采用布地奈德雾化吸入治疗,方法为0.5mg/ml布地奈德加入1ml生理盐水中,放入压缩雾化器中雾化吸入,bid。支气管哮喘组激发后以生理盐水雾化吸入,对照组雾化生理盐水作为对照。上述处理共持续7天。各组于末次诱发哮喘后24小时内麻醉,分离右侧股动脉,留取血标本,取血清-70℃保存,备做酶联免疫。右肺组织灌入4%多聚甲醛至右肺膨胀,然后结扎右支气管,固定,取右肺中叶组织,备做免疫组化。4指标的检测4.1 IL-12与IL-25的检测应用ELISA方法检测大鼠血清IL-12及IL-25的含量,其操作步骤以试剂说明书进行。4.2免疫组化法检测哮喘肺组织中NF-κB与MMP-9的表达采用SP9001通用二步法试剂盒检测肺组织中NF-κB与MMP-9的表达,光镜观察,阳性部位呈棕黄色。设定阳性比率,并行统计学处理。结果:1哮喘大鼠模型鉴定模型组大鼠在吸入OVA后不久即出现躁动不安,搔抓颜面皮毛等过敏症状,继而大鼠俯伏不动,出现呼吸急促,呼吸困难(明显的腹式呼吸),部分大鼠可闻及响亮的呼气相喘鸣音,口鼻流出粘液,严重者可出现呼吸不规则,反复激发后大鼠毛色失去光泽,精神不振和反应迟钝。2酶联免疫检测结果2.1血清IL-25水平的变化哮喘组血清IL-25含量是(40.71±3.87)pg/ml,激素治疗组血清IL-25含量是(30.43±1.48)pg/ml,对照组血清IL-25的含量是(25.96±1.74)pg/ml,三者比较有统计学意义(P<0.05)。并且哮喘组血清IL-25含量(40.71±3.87)pg/ml高于激素治疗组(30.43±1.48)pg/ml,两者比较有统计学差异,明显高于对照组(25.96±1.74)pg/ml,两者比较有统计学差异(P<0.05),激素治疗组与对照组之间没有显著性差异(P>0.05)。2.2血清IL-12水平的变化哮喘组血清IL-12含量是(25.91±3.20)pg/ml,激素治疗组血清IL-12含量是(29.42.±2.43)pg/ml,对照组血清IL-12的含量是(33.71±2.69)pg/ml,三者比较有统计学意义(P<0.05)。并且哮喘组血清IL-12含量(25.91±3.20)pg/ml较激素治疗组降低,两者比较有统计学差异(P<0.05),较对照组明显降低,有统计学差异(P<0.05),激素治疗组与对照组之间有统计学意义(P<0.05)。3各组大鼠组织标本的制备2.3血清IL-25/ IL-12比值变化模型组血清中IL-25/ IL-12比值与正常组相比明显升高,差异有显著性(P<0.01);经激素治疗后,血清中IL-25/ IL-12比值与模型组相比降低,与正常组相比升高,差异亦均有显著性(P<0.01)。3切片结果的鉴定3.1光镜结果哮喘大鼠细支气管痉挛、收缩呈菊花状,管腔内有分泌物,细支气管、小血管周围见明显炎性细胞浸润,以嗜酸细胞、淋巴细胞为主,亦见中性粒细胞;肺泡间隔增宽,有纤维化,大量淋巴细胞、巨噬细胞浸润。治疗组大鼠细支气管无明显痉挛、收缩,管腔内无分泌物,管壁周围炎性细胞浸润明显减少,肺泡壁间隔无增宽。3.2免疫组化检测结果NF-κB和MMP-9在模型组、激素治疗组和正常对照组大鼠肺组织中均有阳性表达,并主要在细胞核表达,少量在细胞浆表达。3.2.1肺组织内NF-κB表达的变化模型组肺组织内NF-κB的表达与正常组相比明显升高,差异有显著性(P<0.01);经激素治疗后,大鼠肺组织内NF-κB的表达与模型组相比降低,与正常组相比升高,差异均有显著性(P<0.01)。3.2.2肺组织内MMP-9表达的变化模型组肺组织内MMP-9的表达与正常组相比明显升高,差异有显著性(P<0.01);经激素治疗后,大鼠肺组织内MMP-9的表达与模型组相比降低,与正常组相比升高,差异均有显著性(P<0.01)。4各指标之间的相关性应用Spearman等级相关法分析发现:IL-25与IL-12之间存在负相关(r=-0.754, P<0.01),IL-25与NF-κB之间存在正相关(r=0.812, P<0.01),IL-12与NF-κB之间存在负相关(r=-0.747, P<0.01),MMp-9与与NF-κB之间存在正相关(r=0.797, P<0.01)。结论:1哮喘时,Th1/Th2免疫平衡破坏,T细胞向Th2方向偏移,Th2优势反应在哮喘发病机制中有重要意义。2哮喘组大鼠NF-κB支气管肺组织中表达明显高于对照组,说明哮喘气道炎症及重塑过程中NF-κB具有重要作用。3哮喘组大鼠NF-κB活性增强,IL-25水平上升,IL-12水平下降,IL-25/IL-12升高,相关分析示:NF-κB与Th2类细胞因子IL-25成正相关,与Th1类细胞因子IL-12成负相关。4哮喘组大鼠NF-κB活化与MMP-9支气管肺组织中表达明显高于对照组,支气管黏膜增生肥厚明显,表明两者参与气道重塑的过程,相关分析示两者存在正相关。5布地奈德作为治疗哮喘的有效药物,可以抑制NF-κB活性,使IL-25水平降低,IL-12水平升高,改善减轻气道炎症。使MMP-9水平降低,改善气道重塑。可作为治疗哮喘的首选。为临床治疗哮喘提供理论依据。更多还原

【Abstract】 Objective: Bronchial asthma is recognized as one of the world’s four major chronic medical profession. The leading cause of death was listed as the most. Now, with people’s lives change, the incidence rate increase every year. The clinical manifestations is sudden onset recurrent wheezing, difficulty breathing, chest tightness and coughing. The pathological basis of bronchial asthma is airway inflammation and airway remodeling. In this study, through the establishment of asthma model, we observe inhaled budesonide in asthmatic rat the LI-25/ IL-12 ratio change and explore the relationship between the immune imbalance and the expression of NF-κB dynamic in asthma. Observation of inhaled budesonide airway NF-κB, MMP-9 expression in asthmatic rat, we explore relationship between airway remodeling and NF-κB.Methods:1 Animals and drugsWe selected 30 wistar mouse, provided by the Experimental Animal Center of Hebei Province (experimental animal quality certification number: 90301 );Budesonid produced by AstraZeneca Pharmaceuticals; Immunohistochemistry kit, one anti-cox2 rabbit anti-IgG polyclonal antibody, purchased in boshide company, SP9001 kit and DAB reagent purchased in Bio-Technology ,Beijing T-shirt. Enzyme immunoassay kit purchased in the United States RB; other experimental drugs provide by immunity teaching of the Hebei Medical University.2 Model preparation and experimental groupsthe law use random numbers and 30 male Wistar rats were randomly divided into three groups:normal control group,asthma model group, budesonide-treated group,10 in each group.Preparation of asthma model group, two-step:sub-sensitized and challenged. In addition to the normal control group, the other two groups were injectioned antigen solution 1ml (including egg protein, 100mg, aluminum hydroxide powder 100mg) by intraperitoneal sensitized on 1 day, 8 day, 15 day three times. and normal control group with equal of saline to sensitize; the 21 days, the rats were placed not completely closed self-made plexiglass box, asthma model group and the budesonide treatment group were Sucked in 1% ovalbumin (OVA) solution by the air compression pump spray Fog excitation,once a day,each20 minutes,continuously 7 days.The budesonide treatment group after excitation are used to budesonide inhalation treatment ,method 0.5mg/ml adding to 1ml saline. Budesonide compressed into the inhalation nebulizer, bid. Bronchial asthma group inhalation the saline after excitation. normal control group were stimulated with saline for the treatment. This last 7 days.3 The preparation of tissue samplesIn each groups, in the Last 24 hours after induced asthma, intraperitoneal injection of anesthesia, the right femoral artery get blood samples. the serum -70℃preservation, prepared to do enzyme-linked immunosorbent . ligation of the left lung, the right lung was slowly poured into 4% paraformaldehyde for the right lung inflated. then ligating of the right bronchus and placing the fixedan.taking right middle lobe organization prepared to do immunohistochemistry.4 Target detection4.1 IL-12 and IL-25 detectionApplication of ELISA to detect serum IL-12 and IL-25 levels, their manual steps carry out the reagen.4.2 TO detect NF-kB and MMP-9 expression in the lung tissue with immunohistochemistrySP9001 Universal two-step method using to detect NF-kBand MMP-9 expression in the lung tissue, light microscopic observation, the positive part of brownish yellow. Set-positive rate, parallel statistically.Results: 1 Identification of rat model of asthmaThe model group rats after inhalation of OVA appear restless, scratch facial fur and other allergic symptoms, then bowed down and not move , shortness of breath, difficulty breathing ( the notable abdominal breathing), and some rats were audible and louddly expiratory wheezing, nose and mouth out of mucus,severe cases,irregular breathing could occur.Repeatedly stimulated, fur dull, lassitude and unresponsiveness.2 Enzyme-link ed immunosorbent assay results2.1 The serum IL-25 levelsThe serum IL-25 concentration (40.71±3.87) pg/ml in asthma group, The serum IL-25 concentration (30.43±1.48) pg/ml in hormone therapy group, The serum IL-25 concentration (25.96±1.74) pg/ml in control group ,three groups have more statistically significant (P <0.05). And asthma group serum IL-25 levels(40.71±3.87) higher than the hormone therapy group (30.43±1.48) pg/ml, their have more significant differences. Asthma group serum IL-25 levels (40.71±3.87) significantly higher (25.96±1.74) pg /ml than the control group, their have more significant difference (P <0.05), The hormone therapy group and the control group was no significant difference. (P> 0.05).2.2 The serum IL-12 levelsThe serum IL-12 concentration (25.91±3.20) pg/ml in asthma group, The serum IL-12 concentration (29.42.±2.43) pg/ml in hormone therapy. serum IL-12 concentration (33.71±2.69) pg/ml in control group, three groups have more statistically significant (P <0.05). And asthma group serum IL-12 levels (25.91±3.20) pg/ml lowwer than the hormone therapy group,Their have a statistically significant difference (P <0.05), asthma group serum IL-12 levels (25.91±3.20) pg / ml significantly lower than the control group, their have significant difference (P <0.05),the hormone therapy and the control group were statistically significant (P <0.05).2.3 Serum IL-25 / IL-12 ratio of changeCompared with the normal group, asthma group serum IL-25 / IL-12 ratio was significantly higher, the difference was significant (P <0.01); After hormone treatment, serum IL-25 / IL-12 ratio, compared with asthma group, ratio decreased; compared with the control group increased, the difference also was significant (P <0.01).3 Identification of biopsy results3.1 Light microscopyAsthmatic rat bronchioles had spasm,contraction, lumen containing secretions,bronchioles, the small blood vessels around the apparent infiltration of inflammatory cells,including eosinophils, lymphocyte predominance and neutrophils;alveolar interval widened, fibrosis, a large number of lymphocytes, macrophages infiltration. Treated rats had no significant bronchiole spasm, contraction, non-intraluminal secretions, wall significantly reduced infiltration of inflammatory cells ,without interval widened.3.2 Immunohistochemistry resultsNF-κB and MMP-9 in the asthmiatic group, hormone therapy group and normal control rats were positive expression in lung tissue and were mainly expressed in the nucleus, a small amount of expression in the cytoplasm.3.2.1 Lung tissue NF-κB expressionAsthmatic group, the lung tissue expression of NF-κB was significantly higher compared with the normal group, the difference was significant (P <0.01); After hormone treatment, the rat lung tissue expression of NF-κB compared with Asthmatic group lower , when compared with the normal group increased, the differences were significant (P <0.01)3.2.2 Lung tissue MMP-9 expressionAsthmatic group,The lung tissue expression of MMP-9 was significantly higher compared with the normal group,the difference was significant (P <0.01); After hormone treatment, the rat lung tissue expression of MMP-9 compared with the Asthmatic group reduced,compared with the control group increased, the differences were significant (P <0.01).4 The correlation between the various indicatorsApplication of Spearman Rank Correlation analysis showed that:IL-25 and IL-12 were negative correlation (r =- 0.754, P <0.01), IL-25 and NF-κB were positive correlation (r = 0.812, P < 0.01), IL-12 and NF-κB were negative correlation (r =- 0.747, P <0.01), MMP-9 and NF-κB were positive correlation (r = 0.797, P <0.01).Conclusion:1 The immune balance of Th1/Th2 was broken,and it migrant to Th2 direction.Th2 dominant reaction play an important role in the pathogenesis of asthma.2 The percentage of NF-κB in asthma mice was notable higher than that of the control group.It hint that plays an important role in the process of the airway inflammation and remodeling of asthma.3 In asthma group.the positive percentage of NF-κB and the level of IL-25 increase signicantly,but the level of IL-12 decreased, the ratio of IL-25/IL-12 increase, correlation analysis showed: NF-κB and IL-25 were positively correlated:NF-κB and IL-12 were negative correlation.4 Asthma, NF-κB activation and MMP-9 expression in bronchopulmonary tissues were significantly higher than control group.Bronchial mucosal hyperplasia hypertrophy significantly, indicating that both participate in the process of airway remodeling,correlation analysis showed a positive correlation between the two.5 Budesonide as the effective drug treatment of asthma can inhibit NF-κB activity,so that it is IL-25 levels decrease, IL-12 levels increased, and reduce airway inflammation;Budesonide also can reduce the level of MMP-9 and improve airway remodeling.Can be used as first choice for treatment of asthma and provide a theoretical basis for the treatment of asthma.更多还原