【作者】 孙妍；

【导师】 韩秀珍； 王玉林；

【作者基本信息】 山东大学 ， 儿科学， 2009， 博士

【摘要】 气道重塑是支气管哮喘(简称哮喘)的特征性病理改变,表现为气道平滑肌增生肥大,粘液腺增多,网状基底膜增厚,此外还表现为气道粘膜下层和(或)粘膜固有层血管数目和血管面积的增加,此现象称为“血管生成”。血管生成是支气管哮喘气道重塑的重要组成部分,伴随气道重塑发生发展。新生血管是炎症细胞进入气道壁的门户,促进气道壁增厚,进而引起气流阻塞和气道高反应性。越来越多的研究表明,哮喘患者肺组织的血管数目和血管面积较正常人群显著增加,血管生成在支气管哮喘的进展中发挥重要作用。近年来,对于哮喘患者血管生成的组织病理学有了进一步的了解,但是对血管生成机制的认识还很欠缺。哮喘肺组织血管生成是气道在低氧条件下炎症反应反复刺激的结果,细胞因子在其中发挥着重要作用。炎症区域分子-1(found in inflammatory zone 1,FIZZ1)又称为抵抗素样分子α(resistin-like moleculeα,RELMα)是近年来发现的与肺部疾病密切相关的炎症因子。FIZZ1和Ym-1同为选择性活化巨噬细胞的重要标记物。炎症反应时期,FIZZ1蛋白家族在肺组织表达水平显著增加。FIZZ1刺激肺血管平滑肌细胞和肺内皮细胞增殖,促进血管内皮生长因子(vascularendothelialcell growth factor,VEGF)表达增高。VEGF是血管生长关键的影响因子,增加血管通透性,并直接刺激血管内皮细胞的分化增殖和迁移,促进血管构建及生长。VEGF在低氧环境下的表达受缺氧诱导因子-1α(hypoxia inducible factor 1α,HIF-1α)的调控。HIF-1α是存在于哺乳动物体内的氧特异感受器,在缺氧状态下的信号转导中有重要作用。HIF-1α促进下游基因VEGF的表达,增加血管生成,满足机体代谢需要。同时,血管生成供给炎症细胞营养,促进炎症反应发展。研究提示,FIZZ1、HIF-1α和VEGF在血管生成中有重要作用,目前哮喘肺组织血管生成的研究中,上述因子的作用鲜有报导。吸入型糖皮质激素是控制哮喘的一线用药,可减轻气道炎症反应,有抑制血管生成的报道。作为治疗哮喘的激素类药物之一,布地奈德影响哮喘血管生成的作用机制研究很少。本研究通过卵清蛋白致敏和激发小鼠建立哮喘模型,观察哮喘小鼠肺组织的血管生成变化,以及细胞因子(FIZZ1和VEGF)在哮喘肺组织血管生成中的作用;并通过雾化吸入布地奈德治疗慢性哮喘小鼠,观察糖皮质激素对肺组织血管生成的影响和HIF-1α、VEGF蛋白表达的变化,探讨布地奈德对哮喘小鼠血管生成的作用机制。第一部分:炎症区域分子-1参与哮喘小鼠肺组织血管生成目的:血管生成是支气管哮喘气道重塑的重要组成部分,伴随气道重塑发生发展。血管生成在哮喘的进展中发挥重要作用,其相关机制成为新近研究的热点。FIZZ1是近年发现的与肺部疾病密切相关的炎症因子。FIZZ1刺激肺血管平滑肌细胞和肺内皮细胞的增殖,促进VEGF表达。VEGF是血管生长的关键因子,增加血管通透性,并直接刺激血管内皮细胞的分化增殖和迁移,促进血管的构建及生长。目前尚未见到有关FIZZ1在哮喘小鼠肺组织血管生成中作用及机制的相关报道。本实验中,我们建立哮喘小鼠模型,旨在观察肺组织血管生成的变化,探讨FIZZ1、VEGF蛋白与血管生成的相关性。方法:1.实验动物及动物分组:雌性BALB/c小鼠,体重20g-24g,6-8周龄。小鼠随机分为6组(n=10):A组:哮喘模型卵清蛋白(ovalbumin,OVA)雾化刺激7天组:B组:哮喘模型OVA雾化刺激14天组;C组:哮喘模型OVA雾化刺激28天组;D组:健康对照磷酸盐缓冲液(phosphate buffered saline,PBS)雾化刺激7天组;E组:健康对照PBS雾化刺激14天组:F组:健康对照PBS雾化刺激28天组。D组、E组和F组分别为A组、B组和C组的健康对照。2.建立哮喘小鼠模型:A、B、C组小鼠第1天和第14天分别腹腔注射0.2ml OVA致敏剂溶液,第21天开始予OVA雾化刺激,每日30分钟,建立哮喘小鼠模型。健康对照组以PBS代替OVA致敏并雾化刺激。3.肺组织标本留取:雾化刺激7天后,麻醉处死A组和D组小鼠:雾化刺激14天后,处死B组和E组小鼠;雾化刺激28天后,处死C组和F组小鼠:留取肺组织,置于4%多聚甲醛中固定。4.苏木素&伊红染色观察肺组织病理学改变。5.免疫组织化学染色,观察血管生成和FIZZ1、VEGF蛋白的表达。6.统计学分析。结果:1.哮喘模型组小鼠较健康对照组小鼠肺组织炎症反应明显,气道和血管周围炎症细胞增多,气道上皮部分脱落。哮喘模型组气道周围炎症反应积分随OVA雾化刺激时间的延长较健康对照组小鼠显著增加。2.哮喘模型组小鼠较健康对照组小鼠肺组织血管生成增多,血管面积百分比增加,OVA激发7天后达到高峰,14天后血管面积减少,28天后降低至正常水平。3.FIZZ1在哮喘小鼠表达明显增高,OVA激发7天时达到高峰,14天后表达降低,28天后持续降低,但仍高于正常水平。FIZZ1在健康对照组小鼠肺组织低表达。4.VEGF在哮喘小鼠较健康对照组小鼠表达增高,OVA激发7天时显著高表达,14天后表达降低至正常水平,28天后持续降低,表达水平与健康对照组小鼠无差异。5.血管面积与FIZZ1、VEGF表达均呈现正相关。而且,FIZZ1与VEGF的表达具有显著正相关性。结论:哮喘小鼠肺组织血管生成增多,FIZZ1和VEGF表达增加,FIZZ1调控VEGF表达增高是哮喘小鼠肺组织血管生成增多的可能机制。第二部分:雾化吸入布地奈德对慢性哮喘小鼠缺氧诱导因子-1α及血管内皮生长因子表达的影响目的:血管生成在慢性哮喘的进展中发挥重要作用,能增加炎症细胞浸润从而促进气道壁增厚,引起气道高反应性。目前,肺组织血管生成机制的研究还处于早期阶段。HIF-1α在缺氧状态下的信号转导中有重要作用,能刺激下游基因VEGF的表达,增加血管生成。VEGF是血管生成和血管渗透性的基本调节因子,在血管生长过程中具有重要作用。吸入型糖皮质激素(布地奈德)是控制哮喘的一线药物。目前有关糖皮质激素影响哮喘血管生成机制的研究很少,尚未见到有关布地奈德对慢性哮喘小鼠肺组织HIF-1α和VEGF表达影响的报道。本研究旨在建立慢性哮喘小鼠模型,探讨布地奈德影响哮喘肺组织血管生成的作用机制。方法:1.实验动物及分组:雌性BALB/c小鼠,30只,体重20g-24g,6-8周龄。将小鼠随机分为3组(n=10):哮喘模型组,治疗组和健康对照组。2.建立慢性哮喘小鼠模型:哮喘模型组小鼠第1天和第14天分别腹腔注射0.2mlOVA致敏剂溶液,第21天开始至实验结束OVA雾化刺激,每日30分钟,建立慢性哮喘小鼠模型;健康对照组以PBS代替OVA致敏和雾化刺激;治疗组小鼠造模过程同哮喘模型组,第28天开始每日雾化吸入布地奈德150μg/m~2,1小时后再予OVA雾化刺激维持哮喘状态。3.肺组织标本留取:治疗20周后,麻醉处死哮喘模型组、治疗组和健康对照组小鼠,留取肺组织,部分置于4%多聚甲醛固定,部分置于液氮中保存。4.苏木素&伊红染色观察肺组织病理学改变。5.Masson三色染色法观察肺组织胶原纤维生成情况。6.免疫组织化学染色,观察肺组织血管生成、HIF-1α和VEGF蛋白的表达。7.蛋白印记分析(Western blot)技术检测小鼠肺组织HIF-1α和VEGF蛋白的表达。8.统计学分析。结果:1.哮喘模型组小鼠较健康对照组小鼠肺组织炎症表现明显,气道和血管周围大量炎症细胞浸润,部分气道上皮脱落,胶原纤维明显增多,气道周围炎症反应积分和肺间质纤维化积分显著增加。治疗组与哮喘模型组比较,小鼠肺组织炎症细胞浸润减少,气道壁结构相对完整,胶原纤维减少,炎症反应积分和纤维化积分显著降低。2.哮喘模型组小鼠较健康对照组小鼠肺组织血管生成增多,血管面积百分比增加。治疗组与哮喘模型组比较,肺组织血管面积明显减少。3.哮喘模型组小鼠较健康对照组小鼠肺组织HIF-1α蛋白表达显著增加。布地奈德雾化吸入治疗后,肺组织HIF-1α蛋白表达明显降低。4.哮喘模型组小鼠较健康对照组小鼠肺组织VEGF蛋白表达显著增加。布地奈德雾化吸入治疗后,肺组织VEGF蛋白表达明显降低。5.肺组织血管面积与HIF-1α、VEGF蛋白表达均呈正相关。HIF-1α与VEGF蛋白表达呈现显著正相关。结论:雾化吸入布地奈德能抑制慢性哮喘小鼠血管生成,降低HIF-1α和VEGF的表达。血管面积与HIF-1α、VEGF蛋白表达均呈正相关,提示HIF-1α/VEGF通路是布地奈德抑制慢性哮喘小鼠肺组织血管生成的可能机制。第三部分条件免疫反应对慢性哮喘小鼠的作用研究目的:条件免疫反应(conditioned immune response,CIR)又称条件反射性免疫反应,是根据经典巴甫洛夫条件反射的训练方式,将新异刺激与能够引起机体免疫反应的非条件刺激相结合,经训练强化后,单独呈现条件刺激实现对免疫系统的影响。越来越多的研究表明,条件免疫反应和支气管哮喘的发作存在密切联系。本实验建立慢性哮喘小鼠模型,以声音为条件刺激,以布地奈德和沙丁胺醇联合雾化吸入为非条件刺激,建立条件免疫反应,观察条件免疫反应对于慢性哮喘小鼠呼吸频率的影响及肺组织病理学改变。方法:1.实验动物及分组:雌性BALB/c小鼠90只,体重20-24g,6-8周龄。将小鼠随机分为6组(n=15):A组:条件免疫反应组,以声音为条件刺激,布地奈德和沙丁胺醇联合雾化吸入为非条件刺激,建立条件免疫反应,治疗20周。B组:常规治疗组,联合雾化吸入布地奈德和沙丁胺醇,1次/日,共20周。C组:减量治疗组,联合雾化吸入布地奈德和沙丁胺醇,1次/日,共7天,之后1次/周,共20周。D组:声音刺激组,仅给予声音刺激,1次/日,共20周。E组:哮喘对照组。F组:健康对照组。2.建立慢性哮喘小鼠模型:A组、B组、C组、D组和E组小鼠于第1天和第14天分别腹腔注射0.2mlOVA致敏剂溶液,第21天开始至实验结束予OVA雾化吸入,1次/日,每次30分钟,建立慢性哮喘小鼠模型。健康对照组小鼠,PBS替代OVA致敏和雾化激发。3.记录各组小鼠呼吸频率的变化。4.苏木素&伊红染色观察肺组织病理学改变。5.Masson三色染色法观察肺组织胶原纤维生成情况。6.统计学分析。结果:1.A组(条件免疫反应组)、B组(常规治疗组)较E组(哮喘对照组)小鼠呼吸频率明显降低,A组和B组之间无显著性差异。C组(减量治疗组)、D组(声音刺激组)与E组相比,呼吸频率无明显差异。E组较F组(健康对照组),呼吸频率显著增加。2.A、B组与E组相比较,小鼠肺组织炎症细胞浸润和气道上皮损伤明显改善,气道周围炎症反应积分明显降低。C组、D组与E组相比,炎症反应积分无明显差异。E组与F组相比,炎症反应明显,气道和血管壁周围有大量炎症细胞浸润,气道上皮细胞部分脱落,气道周围炎症反应积分显著增高。3.A、B组与E组相比较,小鼠肺间质胶原纤维显著减少,肺间质纤维化积分明显降低。C组、D组与E组相比,纤维化积分无明显差异。E组与F组相比,肺间质胶原纤维明显增多,肺间质纤维化积分显著增高。结论:以声音为条件刺激,以布地奈德和沙丁胺醇联合雾化吸入为非条件刺激,建立条件免疫反应,可以减轻炎症反应,抑制纤维化,对慢性哮喘小鼠具有治疗作用。更多还原

【Abstract】 The airway remodeling is a distinctive feature of asthma,characterized by hypertrophy and hyperplasia of airway smooth muscle,increase of mucous gland, thickening of the reticular basement membrane,and qualitative and quantitative changes of airway blood vessel.The increase in the number and size of vessel contributes to the thickening of airway wall,which in turn leads to critical narrowing of bronchial lumen.More recent studies found an increase in the total number of vessels in patients with asthma when compared with control subjects.Angiogenesis plays an important role in airway remodeling in asthma,while the mechanisms underlying this process are not fully elucidated.Some cytokines are involved in airway allergic inflammation,and also in the formation of vessels,such as found in inflammatory zone 1(FIZZ1),vascular endothelial growth factor(VEGF)and hypoxia-inducing factor 1α(HIF-1α).FIZZ1,a novel cysteine-rich secreted protein,is able to promote proliferation and migration of pulmonary endothelial cells,and is able to upregulate the expression of VEGF.VEGF is known to be one of the most important cytokines in the formation process of vessels,and it is the important down-stream cytokine of HIF-1a.HIF-1a is an essential transcription factor,whose expression and activity are tightly regulated by cellular oxygen concentration.In hypoxia,HIF-1a can increase VEGF expression and induce angiogenesis to satisfy the oxygen requirement.According to these reports, FIZZ1,VEGF and HIF-1αare supposed to play a key role in angiogenesis of asthma.Inhaled corticosteroid such as budesonide is the effective drug for asthma, alleviating airway allergic inflammation and reducing the thickness of the basement membrane.To date,there are some studies on the effects of inhaled corticosteroid in angiogenesis,while its mechanisms of the anti-angiogenic effect in asthma are to be elucidated.In this study,murine models are established by ovalbumin(OVA) sensitization and repetitive challenge.Then we are to investigate the changes of airway angiogenesis,the expression of FIZZ1 and VEGF in mouse lung tissues,and further the effects and possible mechanism of FIZZ1 in angiogenesis of asthma in PartⅠ.In PartⅡ,we are to establish murine models of chronic asthma,and to investigate the effects of budesonide on airway angiogenesis,and also the relationship between angiogenesis and HIF-1α,VEGF expression.PartⅠFound in inflammatory zone 1 influences angiogenesis in murine models of asthmaObjectiveAngiogenesis plays an important role in airway remodeling in asthma,while the mechanisms underlying this process are not fully elucidated.Found in inflammatory zone 1(FIZZ1),a novel cysteine-rich secreted protein,is able to promote proliferation and migration of pulmonary endothelial cells,and is able to upregulate the expression of vascular endothelial growth factor(VEGF),which is one of the most important cytokines in the formation of vessels.In this study,the role of FIZZ1 and VEGF in angiogenesis of asthma is to be investigated.MethodsMurine models of asthma were established by ovalbumin(OVA) sensitization on days 1 and 14 and OVA challenge beginning on day 21.Mice were divided into six groups as follows(n = 10):OVA-challenged mice for 7 days,14 days,and 28 days respectively,and healthy mice challenged with PBS for 7 days,14 days,and 28 days respectively.The expression of FIZZ1,VEGF,and von Willebrand factor (vWF)-stained vascular area were measured.Histologic examination was also performed on airway inflammation.Results1.There were obvious allergic inflammation in murine models of asthma,and the inflammation scores were increased significantly compared with those of healthy mice.2.vWF-stained vascular area was largely increased after 7-day OVA challenge in models of asthma when compared with that of healthy mice,reduced from day 14 of OVA challenge,and was reduced to the normal levels after 28-day challenge.3.Levels of FIZZ1 were increased significantly in models of asthma,reached the peak by 7-day OVA challenge,declined on day 14,and were reduced further after 28-day OVA challenge.4.VEGF expression was increased after 7-day OVA challenge in models of asthma when compared with that of healthy mice.And VEGF expression was reduced to the normal level after 14-day OVA challenge,and was reduced further after 28-day challenge.5.Vascular area was positively correlated with expression of both FIZZ1 and VEGF.And there was positive correlation between FIZZ1 expression and VEGF expression.ConclusionThere is obvious angiogenesis in lung tissues of murine models of asthma.FIZZ1 expression was increased,and was significantly correlated with vascular area and VEGF expression,suggesting that FIZZ1/ VEGF passageway plays a key role in angiogenesis of asthma. PartⅡEffects of budesonide on the expression of hypoxia inducible factor-1αand vascular endothelial growth factor in murine models of chronic asthmaObjectiveAirway inflammation in asthma is usually accompanied with formation of new blood vessels.Angiogenesis plays an important role in airway remodeling in asthma. Hypoxia-inducing factor 1α(HIF-1α) is an essential transcription factor in hypoxia, whose expression and activity are tightly regulated by cellular oxygen concentration. HIF-1αcan increase VEGF expression and induce angiogenesis to satisfy the oxygen requirement in hypoxia.VEGF is known to be one of the most important cytokines in the vessel formation.And inhaled corticosteroid,such as budesonide,is the effective drug for asthma,while the mechanisms of its effects on angiogenesis are not fully elucidated.In the present study,we applied inhaled budesonide to murine models of chronic asthma,and to elucidate the effect of budesonide on airway angiogenesis and on the expression of HIF-1αand VEGF.MethodsWe established murine models as follows:mice were sensitized on days 1 and 14 by ovalbumin(OVA) and challenged with OVA beginning on day 21.Mice were divided into three groups(n=10):models of asthma,budesonide-treated mice (100ug/kg.d).and healthy mice.The expression of HIF-1α,VEGF,and von Willebrand factor(vWF)-stained vascular area were measured using immunohistochemistry and Western blot,and airway histologic changes were also examined by hematoxylin & eosin staining and Masson Trichrome staining.Results1.In lung tissues of models of asthma,vascular area was increased,and was reduced significantly after administration of budesonide.2.HIF-1αexpression was elevated in models of asthma when compared with that of healthy mice,which was significantly reduced in budesonide-treated mice.3.VEGF expression was also elevated in models of asthma when compared with that of healthy mice,and was reduced in budesonide-treated mice.4.Vascular area was positively correlated with expression of both HIF-1αand VEGF.And there was positive correlation between HIF-1αexpression and VEGF expression.5.There were obvious allergic inflammation and collagen deposition in murine models of chronic asthma,and the inflammation scores and fibrosis scores were increased significantly,which were alleviated in budesonide-treated mice.ConclusionBudesonide inhibits angiogenesis,also the HIF-1αand VEGF expression in asthma. Vascular area was positively correlated with expression of both HIF-1αand VEGF.And there was positive correlation between HIF-1αexpression and VEGF expression.The results indicate that HIF-1α/VEGF passageway plays an important role in the anti-angiogenic effects of budesonide. PartⅢThe effects of conditioned immune response in murine models of chronic asthmaObjectiveConditioned immune response is also called the conditioned reflex immunoreaction, pairing a novel conditioned stimulus(CS) with unconditioned stimulus(UCS) that makes immune changes in organism.After CS-UCS pairings,re-exposure of animals to the CS alone results in conditioned immune response usually ascribed to the effects of UCS.Behavioral conditioning has been demonstrated to regulate both humoral and cellular immunity.However,its effects on asthma are not clear.In our experiment,we established murine models of chronic asthma,and applied tone to mice as CS,inhaled budesonide(BUD) and salbutamol(SAL) as UCS,and observed the effects of conditioned immune response in murine models of chronic asthma.MethodsChronic murine models were established as follows:mice were established by ovalbumin(OVA) sensitization on days 1 and 14 and OVA challenge beginning on day 21.Models of asthma were divided into groups as follows and received different treatments from day 28:In CS+UCS group(A),tone was applied as CS,inhaled budesonide and salbutamol as UCS,concitioned immune response models were established.In daily UCS group(B):mice were inhaled budesonide and salbutamol daily.In low-dose UCS group(C):mice were inhaled budesonide and salbutamol for the first 7days,then once a week.In CS group(D):mice were given tone daily.In OVA group(E):mice were the control models of chronic asthma.The mice in PBS group(F) were sensitized and challenged with PBS instead of OVA as the healthy controls. After 20-week different treatments beginning on day 28,the breath frequency of mice was recorded.Airway histologic changes were also examined by hematoxylin & eosin staining and Masson Trichrome staining.Results1.Breath frequency in the group A(CS+UCS group) and the group B(daily UCS group) was much reduced than that in the group E(OVA group).There was no significant difference between the group A and B.The breath frequency was much increased in the group C(low-dose UCS group),group D(CS group) and group E when compared with that in the group F(PBS group ). 2.Airway allergic inflammation in lung tissues was alleviated in the group A and group B when compared with those in the group E.The inflammation scores were much more in the group C,group D and group E when compared with those in the group F.3.Airway collagen deposition in lung tissues was alleviated in the group A and group B when compared with those in the group E.The fibrosis scores were much more in the group C,group D and group E when compared with those in the group F.ConclusionBehavioral conditioning inhibits airway allergic inflammation and collagen deposition in murine models of chronic asthma,which is concordant with the effect of actual drug administration.更多还原