【作者】 李兰英；

【导师】 李云；

【作者基本信息】 南华大学 ， 儿科学， 2014， 硕士

【摘要】 目的通过建立大鼠慢性哮喘模型，观察卡介苗多糖核酸（polysaccharid nucleicacidfraction of BCG, BCG-PSN）对哮喘大鼠肺组织信号转导及转录活化因子3（SignalTransducer and Activator of Transc, STAT3）及白细胞介素-21（Interleukin, IL-21）表达的影响。方法1.40只清洁健康，约1月龄，体重150-170克的雄性SD大鼠，随机分配为以下四个组别：正常对照组（Control组）、哮喘模型组（Model组）、地塞米松干预治疗组（Dexamethasone，DXM组），布地奈德混悬液干预治疗组（Budesonide，BUD组），每组各10只。2. Model组、DXM组、BCG-PSN组大鼠分别于实验第1天、第15天腹腔注射10﹪卵清蛋白(Ovalbumin，OVA)溶液1毫升，包含免疫佐剂氢氧化铝100毫克，实验第22天，用2﹪卵清蛋白溶液4毫升对大鼠进行雾化吸入激发，每次约20分钟，隔天雾化1次，连续共9周。3.DXM组（0.3mg/kg）、BCG-PSN组（0.45mg/kg）在实验开始后腹腔注射，3次/周，时间共为12周；Control组及Model组给予生理盐水腹腔注射，3次/周，时间共为12周。4.实验第12周末，四组大鼠分别予以水合氯醛腹腔注射麻醉后用肺功能检测气道阻力变化，检测完成后用腹主动脉放血方法处死大鼠，分别取各组左右两侧肺组织，其中左肺组织作实时荧光定量聚合酶连锁反应法（quantitative real-time PCR，qPCR）来检测肺组织STAT3与IL-21mRNA的转录水平，并通过Western Blot检测方法检测大鼠肺组织中STAT3的蛋白表达水平；右侧肺组织通过病理HE染色方法对各组大鼠肺组织进行形态学观察，分析四组大鼠中气道炎症的变化情况。结果1.慢性哮喘大鼠模型的建立：实验第22天开始予以2﹪卵清蛋白溶液雾化吸入后逐渐出现以下哮喘发作症状：面部搔抓、呼吸频率加快、点头呼吸、面部紫绀，大小便失禁等，于实验第12周末予动物肺功能检测其气道阻力变化，结果提示Model组大鼠气道阻力明显增高，与Control组比较差异有统计学意义（P＜0.05）；病理HE染色肺组织切片形态学发现：Model组肺组织中支气管与肺泡周围炎症细胞明显增高，嗜酸性粒细胞增多，气道内有较多粘性分泌物，支气管管壁增厚导致管腔狭窄，提示慢性哮喘大鼠模型构建成功。2.BCG-PSN干预后大鼠肺组织病理学与气道阻力测定：哮喘大鼠经BCG-PSN干预后病理组织学改变，BCG-PSN组仍可见炎症反应，与Model组比较炎症减轻；与DXM比较，差异不显著；但与Control组比较仍可见炎症反应。四组大鼠均以不同浓度盐酸组胺激发。相同浓度盐酸组胺激发下， BCG-PSN组气道阻力与Model组比较，气道阻力明显下降，有统计学意义(P＜0.05)；与Control组比较，气道阻力仍然增高，有统计学意义(P＜0.05)；与DXM组比较，无统计学意义。3.BCG-PSN干预后肺组织STAT3和IL-21表达量的变化：BCG-PSN干预后哮喘大鼠肺组织STAT3、IL-21mRNA转录较Model组下降，差异均有统计学意义(P＜0.05)；但较Control组明显增高，差异均有统计学意义(P＜0.05)；与DXM比较差异无统计学意义。BCG-PSN干预组STAT3蛋白表达量较Model组比较显著降低，差异有统计学意义(P＜0.05)；但较Control组明显增高，差异有统计学意义(P＜0.05)；与DXM比较差异无统计学意义。结论1.通过OVA致敏，激发，能成功构建大鼠慢性哮喘模型。STAT3、IL-21在大鼠肺组织中高表达，参与了哮喘发病。2.BCG-PSN能降低哮喘大鼠气道高反应性、减轻气道炎症。3.BCG-PSN能抑制哮喘大鼠STAT3、IL-21蛋白表达，提示BCG-PSN的干预作用与下调肺组织STAT3、IL-21的表达有关。更多还原

【Abstract】 ObjectiveIn this article, by observe the expression of Interleukin-21,(Signal Transducer andActivator of Transcription3in lung tissue of rat and chronic asthmaic rat, and to observethe expression changes after use BCG-Polysaccharide and Nucleic Acid injection changes,to provide the basis for the mechanism to further clarify the BCG polysaccharide nucleicacid treatment of asthma in children.Methods1.40male SD rats were randomly assigned to four groups which were clean and healthy, about a month old, weighing150-170g.the four groups were normal control group (Control group), asthma model group (Model group), Dexamethasone intervention group (DXM group), BCG-PSN group), each10.2.the experimental rats in Model group, DXM group, BCG-PSN group were intraperitoneal injected by10%egg albumin (Ovalbumin, OVA) solution of1ml contains100mg of aluminum hydroxide on the first day,the15th days. Then were atomized inhalated with2%ovalbumin solution4ml on the22th day,20minutes eachtime, once two days, lasting for12weeks. The Control group used0.9%sodiumchloride solution instead of ovalbumin in intraperitoneal injection and inhalation.3.From the first day of experiment, in Dexamethasone group,0.5h before aerosolizedOVA, each rat was given0.3mg/kg dexamethasone by means of intraperitoneal injection;In BCG-Polysaccharide and Nucleic Acid injection group, each rat was given0.45mg/kgBCG-Polysaccharide and Nucleic Acid injection by means of intraperitoneal injection.three times for every weeks, lasting twelve weeks.4. On the12th weeks of experiment, four groups of rats were anesthetized by intraperitoneal injection of chloral hydrate,then detected the airway hyper responsiveness.After test the rats were sacrificed with abdominal aorta bleeding methods,everylung tissue was detected by quantitative real-time PCR,to observer the expression of STAT3mRNA and IL-21mRNA in lung tissue, the expression of STAT3protein was detected by Western Blot. At the same time the morphology of lung tissueof rats in each group were observed.Result1.Construction of subacute asthma rat model: From the twenty-two days of theexperiment these following symptoms begin to appear:facial scratching, respiratoryfrequency speed, nodding breathing, facial cyanosis, incontinence.on the twelves weeks the airway hyper responsiveness were detected, The results suggest that airway resistance in asthma model group rats increased significantly, compared with the normal control group were statistically significant (P <0.05);HE staining of lung tissuesections suggest that bronchial and alveolar lung tissue of rats with inflammatorycells increased significantly, eosinophils increased,more viscous secretions, bronchialwall thickening, which showed that the rat model of chronic asthma was established successfully.2.after BCG-PSN intervention in lung tissue of rats with pathology: determination andairway resistance in asthma rats with intervention of BCG-PSN pathological changes, canstill be seen in BCG-PSN group compared with Model group, inflammatory reaction,inflammation; compared with DXM, the difference was not significant; however,compared with the Control group still visible inflammatory reaction. Four groups of ratswere stimulated with different concentrations of histamine hydrochloride, airwayresistance in BCG-PSN group compared with Model group, the airway resistancedecreased, there was statistically significant (P <0.05); compared with Control group, theairway resistance is increased, with statistical significance (P <0.05); compared with DXMgroup, no statistical significance.3.Changes of the expression of STAT3in lung tissue and IL-213after BCG-PSNintervention: BCG-PSN dry lung tissue of asthmatic rats, the prognosis of STAT3IL-21mRNA transcription was lower than in group Model, with statistical significance (P <0.05);compared with Control group, but significantly increased, with statistical significance (P <0.05); and DXM is compared, no significant significance. BCG-PSN intervention group the expression of STAT3is significantly lower than those of Model group, there wasstatistically significant (P <0.05); compared with Control group, but significantlyincreased, with statistical significance (P <0.05); compared with DXM, no statisticalsignificance.Conclusion1.Sensitized by OVA, excitation, to construct model of chronic asthmatic rats. STAT3,high expression of IL-21in lung tissue of rats, involved in asthma pathogenesis.2.BCG-PSN can reduce the airway hyperresponsiveness of asthmatic rats, reduceairway inflammation.3.BCG-PSN can inhibit asthma IL-21protein STAT3, expression in rats, suggestingthat the effects of BCG-PSN associated with the downregulation of expression of STAT3,IL-21in lung tissue.更多还原