【作者】 陈红梅；

【导师】 罗百灵；

【作者基本信息】 中南大学 ， 呼吸内科， 2008， 硕士

【摘要】 目的:本实验通过COPD大鼠模型并予以红霉素干预观察大鼠肺内MMP-9表达水平的变化。通过本实验认识和探讨体内实验红霉素对COPD大鼠肺内MMP-9表达的影响,进一步认识红霉素的抗炎作用,开拓红霉素在COPD的应用价值。方法:健康雄性Wistar大鼠36只(中南大学实验动物学部提供),雄性,白色,SPF级,体重220±20 g。在安静、室温、避强光的环境中自由进食和饮水一周。36只Wister大鼠随机分为3组,每组12只:COPD模型组,第1、14天注射脂多糖(LPS)200ug/100ul,第2～13天、15～30天每日上午在72升(30cm×40cm×60cm)自制有机玻璃箱内被动吸烟,每天上午1次,每次12支,每次持续0.5h。红霉素干预组,造模方法同COPD组,每天熏烟前用红霉素灌胃治疗(剂量100mg/kg,配制成1.25%红霉素口服液),从第17天开始进行,经气管内注入LPS的条件、烟熏条件以及持续时间均同COPD组,给药2周;正常对照组,标准饲养30天,不做任何干预,第1、14天气管内注入生理盐水100ul。大鼠肺功能测定由湘雅医学院病理生理研究所完成,经微机处理计算出FEV0.3/FVC、R1及Cdyn。左肺10%的中性福尔马林灌注固定20min,再浸入福尔马林固定,常规脱水,石蜡包埋,切片,HE染色,以进行病理形态学观察和免疫组化检测。用免疫组化方法检测各组大鼠肺组织MMP-9的表达,逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测各组大鼠肺组织MMP-9 mRNA表达水平。结果:(1)模型组及干预组大鼠渐出现饮食、活动减少,平均体重较正常对照组减轻,后期可见毛发干枯、发黄,可听到喘息音,正常对照组未见以上情况出现。(2)模型组及干预组大鼠FEV0.3/FVC(%)、R1(气道阻力)、Cdyn(动态肺顺应性)均明显低于正常对照组,差异有显著性(均P＜0.05),说明小气道气道阻力增加,存在明显气流受限、阻塞性肺通气功能障碍。(3)光镜下模型组及干预组大鼠肺组织符合慢性支气管炎和阻塞性肺气肿病理改变。(4)模型组肺组织中MMP-9蛋白平均阳性系数显著高于对照组(P＜0.05),干预组与模型组比较阳性系数降低(P＜0.05)。(5)模型组大鼠肺组织中MMP-9mRNA表达量高,其光密度比值显著高于正常对照组(P＜0.05);干预组与模型组光密度比值比较亦降低(P＜0.05)。结论:(1)采用被动吸烟和LPS气道内滴注相结合构建的大鼠COPD模型,能成功复制出COPD大鼠模型。(2)COPD大鼠肺组织中MMP-9水平显著增高,在COPD的发病机制中可能起重要作用。(3)红霉素治疗COPD大鼠从基因水平下调MMP-9的表达抑制炎症细胞的移行而减弱炎症反应。更多还原

【Abstract】 Objective:The experiment observed the changes of lung MMP-9 expression level by erythromycin intervention in rat model of COPD.Our goal is to study and understand the effect of erythromycin on lung MMP-9 expression in vivo experiment of COPD rat,a further understanding of the anti-inflammatory effects of erythromycin,and developing the application of erythromycin in COPD.Methods:36 male rats were averagely divided into 3 groups in random.The COPD model group was established by the way of giving passive smoking for one month and endotracheal injection of LPS.Passive smoking was given once daily(0.5 hour)in a plexiglass box from the second day to the thirteen day and the fifteen day to the thirty day.In the first day and forteen day,LPS(200ug / 100ul)was given in trachea.The Erythromycin intervention group was treated with erythromycin gavage from the seventeenth day for 2 weeks,modelling method was the same with COPD group.The normal control was not given passive smoking. And in the first and forteen day,we gave each rat endotracheal injection of stroke-physiological saline solution instead of LPS.Lung function tests were performed for each rat one month later and we tested FEV0.3/FVC,Rl and Cdyn.Then the rat was put to death and the lung was taken out.Sections of paraffin-embedded left lungs tissue were cut, stained with HE,and processed for pathological observation. Immunohistochemistry was permormed for study the expression of MMP-9.The right lungs tissue was prepared for the examination of the expression of MMP-9 mRNA.Results:(1)COPD and Erythromycin intervention rats gradually emerged diet and activity decreasing,the average body weight mitigated compared with normal control group,in the later period appearing dry and yellow hair and the sound of gasping could be heard. The normal control group was out of this situation.(2)Lung function tests showed that FEV0.3/FVC,Rl and Cdyn values were significantly lower in the COPD and Erythromycin intervention groups than in the normal control group(P＜0.05).(3)The COPD and Erythromycin intervention groups lung tissue showed chronic obstructive bronchitis and emphysema pathological changes by light microscope.(4)Compared with the rats of normal control group,the expression MMP-9 protein increased significantly(P＜0.05)in the COPD rats.The positive coefficience of Erythromycin intervention group was lower than COPD group(P＜0.05).(5)In the COPD group MMP-9 mRNA in lungs increased significantly than normal control group(P＜0.05),the optical density ratio of Erythromycin intervention group was also lower than COPD group(P＜0.05).Conclusion:(1)The rat model of COPD which was established by the way of giving passive smoking for one month and endotracheal injection of LPS was successful,which was proofed by lung function tests and pathological observation.(2)MMP-9 levels in COPD rat lung tissue was significantly higher than normal rats,it plays an important role in the pathogenesis of COPD.(3)Erythromycin treatment of COPD rats reduced MMP-9 protein and mRNA expression levels can inhibit the migration of inflammatory cells and weaken inflammatory response.更多还原