哮喘气道重塑大鼠气道上皮细胞凋亡及布地奈德的干预作用

目的 1)研究哮喘气道重塑大鼠气道上皮细胞的凋亡情况 ；2)观察布地奈德对气道上皮细胞的凋亡和气道重塑的干预作用。材料和方法30只雄性SD大鼠随机分为正常对照组(C组)、哮喘重塑组(A组)、布地奈德治疗组(B组)，每组10只, A组给予致敏并激发12周,建立大鼠气道重塑模型。B组大鼠参照A组，只是激发半小时前，雾化吸入布地奈德1mg。C组大鼠参照A组，只是致敏激发时均用生理盐水代替。肺组织用于HE染色、Masson三色染色、PAS染色,观察气道周围炎症细胞浸润和气道结构改变。 结果 (1)光镜下发现A组明显的气道重塑的病理改变,B组A组较有所减轻(2)透射电镜观察均发现A组明显胶原沉积, B组A组较有所减轻。(3)图像分析结果(Image-Pro Plus 5.0):支气管壁厚度比较，A组 (110.01±13.77) 明显高于C组(77.25±7.56)(P<0.01)， B组(92.85±15.16)明显低于A组(P<0.05)，但高于C组(P<0.05)。平滑肌厚度比较，A组 (45.39±7.51) 明显高于C组(25.72±4.05)(P<0.01)，B组(36.48±4.70)明显低于A组 (P<0.05)，但高于C组(P<0.01)。各组大鼠肺组织胶原百分比比较：A组(12.79±3.75)明显高于C(3.16±0.90)(P<0.01),B组(9.09±1.46)明显低于A组(P<0.05)，但仍明显高于C组(P<0.01) (4) 图像分析免疫组化结果(Image-Pro Plus 5.0软件测阳性细胞数)： cleaved caspase-3阳性细胞数的比较，A组大气道上皮细胞和中小气道上皮细胞分别为[(10.49±2.82)，(9.58±1.86)] 明显高于C组 [(2.86±1.17),(2.44±1.0)](P均<0.01), B组分别为[(10.58±2.51)，(10.65±2.36)] 与A组(10.49±2.82)(9.58±1.86)比较无显著性差异(P均>0.05)，但明显高于C组 (P均<0.01)。结论: 1)哮喘气道重塑大鼠气道上皮细胞表达cleaved caspase－3显著增加，表明气道上皮凋亡在气道重塑组中明显增加。2)BUD不能抑制哮喘大鼠气道上皮细胞发生凋亡。3)布地奈德能部分抑制气道重塑。

A Study on apoptosis in epithelium of asthmatic rat with airway remodeling and the regulation of Budesonide

Objectives：(1)To study the airway epithelial cell apoptosis in asthmatic rat with airway remodeling (2) To investigate the regulation role of BUD(Budesonide) on airway remodeling and apoptosis in asthmatic rat airway epithelium.Methods:Thirty male rats were randomly divided into control group (group C) ,airway remodeling group(group A) and BUD treated group(group B), each group had 10 rats. In the experiment,a rat asthma model was established by the ovalbumin challenged methods. The rats from group A and group B were sensitized by injecting 1ml mixture (contain 1mg OVA and 100mg aluminum hydroxide) at first and eighth days, and challenged with an aerosol of 1% OVA on fifteenth day .The challenge were repeated every other day in the following 12 weeks , 30 minutes one time . group C was injected and challenged by 0.9% sodium chloride. in additional ,The rats of group B were treated 1mg nebulized BUD.An hour after last challenge,the animals were anesthetized and then sacrificed,and the sample of the lung tissue was collected. The left lung tissue was collected and embedded in paraffin ,sectioned. Hematoxylin and eosin staining,Masson's Trichrome stain, Periodic Acide Schiff'staining were performed. Histological changes were detected under light or electronic microscope.immunohistochemistry assays were used to detemined the status of cleaved caspase-3 in airway epithelium.Result:(1)The result of light microscope observation: Compared with group C,light microscope of HE-stained lung tissue showed that there were infiltration of numerous inflammatory cells around the bronchus ,and airway epithelium damaging in the group A. Airway smooth muscle cell proliferation , airway wall thickness ,mucous plugs were found in group A. Enhenced goblet cell hyperplasia in PAS stained sections and excessive collagen deposition in Subepithelial area were found in group A. Pathologic change in BUD treated group were abated in comparasion to group A. yet excessive collagen deposition in Subepithelial area were found in group B (2)The result of TEM:Subepithelial basement membrane thickness and collagen deposition were obviously observed under TEM in both group.A and group B. Infiltration of EOS and AT-II cells damaging were found in group A,and abated in group B.(3)The results of image analysis: compared with the total bronchial wall area(wat), group A(110.01±13.77) was significantly higher than group C(77.25±7.56) (P<0.01), group B (92.85±15.16)was significantly lower than group A (110.01±13.77)(P<0.05)，but higher than group C（P<0.05）.Compared with the smooth muscle area, group A(45.39±7.51) was significantly higher than group C(25.72±4.05) (P<0.01), group B(36.48±4.70) was significantly lower than group A(45.39±7.51) (p<0.05)，but significantly higher than group C（P<0.01）compared with the percentage of collagen deposition area: group A (12.79±3.75) was significantly higher than group C (3.16±0.90)(P<0.01),group B (9.09±1.46) was significantly lower than group A (P<0.05)，but higher than group C (P<0.01)..(4) The results of immunohistochemistry assays: the cleaved caspase-3 imumunoreactive cells increased in rat epithelium in both group A[(10.49±2.82),(9.58±1.86)] and group B [(10.58±2.51)，(10.65±2.36)]in comparasion to that of group C[(2.86±1.17),(2.44±1.0)] (both P<0.01). No significant difference between group A and group B was found( both P>0.05) Conclusion : (1) The apoptosis in the epithelium of asthmatic rat with airway remodeling was significantly increased.(2) BUD treatment fail to inhibit apoptosis in asthmatic rat (3) BUD treatment partly inhibit airway remodeling