【作者】 王宁；

【导师】 安淑华；

【作者基本信息】 河北医科大学 ， 儿科学， 2010， 硕士

【摘要】 背景:支气管哮喘(简称哮喘)是一种呼吸系统常见的慢性疾病,是以气道高反应性、气道慢性炎症及气道重塑为特征的变态反应性疾病。随着社会的发展,其发病率不断上升,特别是儿童哮喘更加明显,给社会及家庭带来长期的负担和压力。因此,哮喘的发病机制和治疗一直是研究的热点,人们对哮喘的认识已经开始在分子-基因水平上探究病因、发病机制及相应的诊断和治疗方法。但是,哮喘的发生是多因素、多基因、多环节相互作用的结果,许多问题有待解决,进一步研究哮喘的发病机制及寻求新的治疗方法成为当前医学界普遍关注和迫切解决的问题。目的:以哮喘大鼠气道模型为基础,通过研究金属蛋白酶组织抑制剂-1(TIMP-1)在气道重塑中的表达情况,并用计算机图像分析软件对气道重塑程度情况进行量化,进而探讨哮喘大鼠模型中TIMP-1与气道重塑的关系;同时应用抗正常T细胞表达和分泌的活性调节蛋白抗体(抗RANTES抗体)对哮喘大鼠进行干预,通过肺组织中TIMP-1表达的变化以及量化的气道重塑情况改变,来探讨抗RANTES抗体对气道重塑的影响,从而为哮喘的临床治疗提供动物实验依据。方法:将36只健康雄性wistar大鼠随机分为三组:哮喘模型组、抗RANTES抗体干预组、正常对照组,每组12只。哮喘模型组和抗RANTES抗体干预组于第1、7、14天腹腔注射10%抗原液1ml(含卵白蛋白100mg、氢氧化铝100mg)进行致敏,正常对照组腹腔注射等量生理盐水致敏。从第21天起将哮喘模型组、抗RANTES抗体干预组大鼠分别置于密闭玻璃罩内,雾化吸入1%的卵白蛋白激发,每次雾化吸入30分钟,隔天1次,共6周,正常对照组用等量的生理盐水进行激发。抗RANTES抗体干预组自雾化激发后1周起即从第28天起每次激发前半小时给予尾静脉注射抗RANTES抗体1ug,哮喘模型组和正常对照组给予尾静脉注射等量生理盐水,隔天1次,共5周。观察三组实验大鼠的一般状况,包括活动、呼吸、神态、毛发等。末次激发结束后24小时用1%戊巴比妥那0.2ml/只腹腔注射麻醉各组大鼠,打开胸腔,取出左肺组织。作HE染色病理图片,光镜观察三组实验大鼠的支气管肺组织的病理改变、用计算机图像分析软件分别测定三组实验大鼠支气管基底膜周长(Pbm)、气道平滑肌面积(WAm)、气道内壁面积(WAi),并用Pbm标准化,分别以WAm/Pbm及WAi/Pbm表示气道平滑肌面积、气道内壁面积,量化气道重塑程度的改变;采用免疫组化方法测定三组实验大鼠肺组织TIMP-1的相对积分光密度的改变,观察TIMP-1的表达情况。结果:(1)三组大鼠的一般状况:①哮喘模型组:雾化吸入卵白蛋白后大部分大鼠出现烦躁不安,搔抓颜面,鼻扇,毛发失去光泽,打喷嚏,口唇发绀,继而大鼠安静不动,弓背,张口呼吸,呼吸加深加快,前肢缩抬,四肢颤抖,口鼻流出粘液,精神不振和反应迟钝,停止雾化后上述表现逐渐缓解。②抗RANTES抗体干预组:第1周每次雾化吸入卵白蛋白后,大部分表现与哮喘模型组相似,而在应用抗RANTES抗体后,虽有烦躁、喷嚏、四肢抓挠,但无明显的呼吸困难、口唇发绀表现。③正常对照组:致敏及任意一次激发后均正常饮食、饮水,精神活泼、反应灵敏、呼吸平稳、毛色光洁,没有呼吸急促、烦躁不安或口周发绀的表现。(2)三组大鼠气道平滑肌的比较:哮喘模型组大鼠气道平滑肌面积WAm/Pbm[(5.78±1.36)μm2/μm]明显大于正常对照组大鼠的气道平滑肌面积WAm/Pbm[(3.35±1.01)μm2/μm] (P<0.05);抗RANTES抗体干预组大鼠气道平滑肌面积WAm/Pbm[(4.12±1.21)μm2/μm]较哮喘模型组大鼠的气道平滑肌面积WAm/Pbm显著降低(P<0.05);抗RANTES抗体干预组大鼠的气道平滑肌面积WAm/Pbm与正常对照组大鼠气道平滑肌面积WAm/Pbm相比无差别(P>0.05)。(3)三组大鼠气道内壁面积的比较:哮喘模型组大鼠气道内壁面积WAi/Pbm[(15.10±3.99)μm2/μm]明显大于正常对照组大鼠的气道气道内壁面积WAi/Pbm[(9.25±2.29)μm2/μm] (P<0.05);抗RANTES抗体干预组大鼠气道内壁面积WAi/Pbm[(10.01±2.69)μm2/μm]较哮喘模型组大鼠的气道内壁面积WAi/Pbm显著降低;抗RANTES抗体干预组大鼠的气道内壁面积WAm/Pbm与正常对照组大鼠的气道内壁面积WAi/Pbm相比无差别(P>0.05)。(4)三组实验大鼠肺组织TIMP-1表达的变化:哮喘模型组大鼠肺组织的TIMP-1表达[积分光密度(1.9580±0.0927)]较正常对照组大鼠肺组织的TIMP-1表达[积分光密度(0.7623±0.1000)]增强(P<0.05);哮喘模型组大鼠肺组织的TIMP-1表达较抗RANTES抗体干预组大鼠肺组织的TIMP-1表达[积分光密度(0.9770±0.0233)]增强(P<0.05);抗RANTES抗体干预组大鼠肺组织的TIMP-1表达与正常对照组大鼠肺组织的TIMP-1表达相比无差别(P>0.05)。结论:(1)哮喘大鼠模型成功;(2)哮喘模型组的大鼠肺组织中TIMP-1的表达高,可能在气道重塑中起到了重要的作用;(3)哮喘大鼠在应用抗RANTES抗体后哮喘症状缓解,气道重塑程度减轻,通过体外干预研究证实抗RANTES抗体可抑制气道重塑,它可能延缓气道重塑的发生。更多还原

【Abstract】 Background:Asthma is an allergic disease,one common disease in chronic illness of respiratory system.Its airway is characteristed by hyperreactivity,chronic inflammation and remodeling.With the development of society,the incidence of asthma upgrades,especially in the children,which brings extended burden and pressure to society and family.The pathogenesis and treatment of asthma are investigative hotspots constantly.Furthermore, realization of asthma in the molecule-gene level has already begun.However, many inflammatory cells and cytokines are correlated with asthma.No current treatment can reduce or prevent asthma in a long period.Further study on pathogenesy of new interference target of asthma has been generally investigated in medical science.Objective:To observe the expression of TIMP-1(tissue inhibitor of metalloproteinase-1) in the lung tissue on the airway remodeling in the rats of the asthmatic model.Measure the airway smooth muscle area and the inner wall area standardized by the basement membrane perimeter with software of computer image analysis in the asthmatic rats model.Quantify the level of the airway remodeling with the smooth muscle area and the inner wall area of the airway.Study the effects of anti-RANTES (regulated on activation normal T cell expressed and secreted) antibody on the airway remodeling,and the new way of prevention of asthma was offered.Method:The rats were randomly divided into three groups:asthmatic model group,anti-RANTES antibody intervention group and normal control group,each group had 12 rats.The rats of asthmatic model group and anti- RANTES antibody intervention group were immunized by intraperitoneal injection of 10% ovalbumin on the first,the seventh and the fourteenth day of the study.The rats of normal control group were given equivalent physiological saline to allergize.From the third weeks,the rats of asthmatic model group and anti-RANTES antibody intervention group were stimulated by aerosol inhalation of 1% ovalbumin (OVA),once of two days,thirty minutes every time,which were repeated six weeks.The rats of normal control group received stimulation with inhalation of physiological saline.From the fourth weeks,the rats of anti-RANTES antibody intervention group were intervened by caudal vein injection of anti-RANTES antibody,one microgramme every time before each stimulation, which were repeated five weeks.The rats of asthmatic model group and the rats of normal control group were caudal vein injection of equivalent physiological saline.To observe the behavior change of the three groups,including the action,the respiration,the appearance and the hairs of rats.The rats were anesthetized with l% pentobarbital when the end of the last stimulation.Pathological slides were prepared from left lung and stained with hematoxylin-eosin.The airway smooth muscle area (WAM),and the inner wall area (WAi) of the airway were measured and standardized by the basement membrane perimeter (Pbm). Expression of tissue inhibitor of metalloproteinase-1 were detected by immunohistochemistry.Results:When the rats were stimulated in the asthmatic model group, they showed restlessness,scratching honor,sneezing,nose fanning,mouth breathing deepenly and quickenly,oral lip cyanochroia,and all the show above- mentioned releasing gradually after the inhalation.The HE stained section of the asthmatic model group showed mucosal edema,air duct thickening,plica to grow in number.Compared with the asthmatic model group,there were mild symptom and inflammation reaction,and decrease in air duct thicken in the anti-RANTES antibody intervention group.The rats of the normal control group showed normal diet and drinking,animation,sensitiveness,breathing stable.The airway smooth muscle area and the inner wall area of the asthmatic model group [WAm/Pbm(5.78±1.36)μm2/μm,WAi/Pbm(15.10±3.99)μm2/μm] was greater than the normal control group[WAm/Pbm(3.35±1.01)μm2/μm, WAi/Pbm(9.25±2.29)μm2/μm](all P<0.05).The airway smooth muscle area and the inner wall area of the anti-RANTES antibody intervention group [WAm/Pbm(4.12±1.21)μm2/μm,WAi/Pbm(10.01±2.69)μm2/μm]was greater than asthmatic model group (all P<0.05),but no statistical significance compared to the normal control group (all P>0.05).The relative integral optical density value of the tissue inhibitor of metalloproteinase-1 in the lung tissues was increased significantly in the asthmatic model group [1.9580±0.0927] than the normal control group [0.7623±0.1000](P<0.05).The relative integral optical density value of the tissue inhibitor of metalloproteinase-1 in the lung tissues in the anti-RANTES antibody intervention group [0.9770±0.0233] was decreased significantly than the asthmatic model group (P<0.05),but no statistical significance compared to the normal control group (P>0.05).Conclusion:The asthmatic rats model were succeeded.The tissue inhibitor of metalloproteinase-1 had relationship with asthma airway remodeling.The anti-RANTES monoclonal antibody leads to decrease of the expression of the tissue inhibitor of metalloproteinase -1 in the lung tissue of asthma rats,lessen asthma attack,inhibit the airway inflammation,delay development of airway reconstitution.The anti-RANTES antibody takes an important role in delaying development of asthma airway remodeling.更多还原