嗜酸粒细胞性支气管炎小鼠模型的建立及嗜酸粒细胞激活对气道反应性的影响

Development of an eosinophilic bronchitis mouse model and the effects of eosinophil activation on airway hyperresponsiveness in eosinophilic bronchitis mice

目的建立嗜酸粒细胞性支气管炎小鼠模型，探讨嗜酸粒细胞激活对气道反应性的影响。方法无特定病原体级雌性BALB/c小鼠60只，按随机数字表法分为多黏菌素B组、生理盐水组、多黏菌素B＋N－甲酰－蛋氨酸－亮氨酸－苯丙氨酸（fMLP）组、生理盐水＋fMLP组。各组小鼠给予对应的点鼻液点鼻，共21 d。其中多黏菌素B组和生理盐水组分别以12 μl的0.5%多黏菌素B和等量生理盐水点鼻，1次/d；多黏菌素B＋fMLP组、生理盐水＋fMLP组除以上对应处理外，分别于第21天点鼻后3 h再予10 μl fMLP（0.05 mg/ml，溶于乙酸中）点鼻1次。末次点鼻后24 h内搜集4组小鼠支气管肺泡灌洗液（BALF）进行细胞分类计数。多黏菌素B组、生理盐水组均于末次点鼻后3 h测定小鼠对乙酰甲胆碱（Ach）的气道反应性（吸气阻力、呼气阻力和肺顺应性）；多黏菌素B＋fMLP组、生理盐水＋fMLP组均于末次点鼻后24 h测定气道反应性。酶联免疫吸附（ELISA）法测定血清、BALF、肺组织匀浆中嗜酸粒细胞过氧化物酶（EPX）、嗜酸粒细胞阳离子蛋白（ECP）、嗜酸粒细胞趋化因子（ECF）水平。肺组织HE染色、变色酸2R染色观察气道周围炎症细胞浸润程度，并在电镜下观察低密度嗜酸粒细胞形态。结果多黏菌素B组BALF细胞总数显著高于生理盐水组[29.50（3.25）×104/ml比15.25（2.25）×104/ml，P〈0.001]，以嗜酸粒细胞最明显[11.76（6.02）×104/ml比0.12（1.08）×104/ml，P〈0.001]；两组小鼠吸气阻力、呼气阻力和肺顺应性差异均无统计学意义。随着注射Ach浓度的逐渐升高，多黏菌素B＋fMLP组吸气阻力、呼气阻力均显著高于其他3组，肺顺应性均显著低于其他3组，血清、BALF、肺组织中EPX、ECP、ECF水平均显著高于其他3组（均P〈0.001）。多黏菌素B、多黏菌素B＋fMLP组肺组织气道周围嗜酸粒细胞明显增多；多黏菌素B组偶见脱颗粒后形成的空泡而成为低密度嗜酸粒细胞，多黏菌素B＋fMLP组见较多激活的低密度嗜酸粒细胞。结论应用多黏菌素B点鼻可建立嗜酸粒细胞性支气管炎小鼠模型；嗜酸粒细胞激活对气道高反应性的发生有重要作用。

ObjectiveTo develop a mouse model of eosinophilic bronchitis, and explore the effects of eosinophil activation on airway hyperresponsiveness.MethodsA total of 60 female specific pathogen free BALB/c female mice were divided randomly into four groups： polymyxin B group, normal saline group, polymyxin B＋ N-methionine leucine phenylalanine （fMLP） group, normal saline＋ fMLP group. All the groups were given corresponding nasal drops for 21 days. The former two groups were given 12 μl 0.5% polymyxin B or normal saline once a day by transnasal administration respectively. Besides the above, the latter two groups were given 10 μl fMLP （0.05 mg/ml, dissolved in acetic acid） once by transnasal administration 3 hours after polymyxin B or normal saline administration on the 21st day. Within 24 hours after the last transnasal administration, cells in bronchoalveolar lavage fluid （BALF） were collected and analyzed by absolute different cell counts. Airway responsiveness （inspiratory and expiratory airway resistance and lung compliance） to acetyl choline （Ach） were measured 3 hours after the last transnasal administration in the former two groups and 24 hours after the last transnasal administration in the latter two groups. Eosinophil peroxidase （EPX）, eosinophil cationic protein （ECP） and eosinophil chemotactic factor （ECF） levels in serum, BALF and lung tissue were tested by emzyme linked immunosorbent assay （ELISA）. HE and Chromotrope 2R staining of lung tissue were used to observe the infiltration of inflammatory cells. The morphology of low-density eosinophils was observed under electron microscope.ResultsThe total cell counts of BALF in polymyxin B group were significantly higher than the normal saline group [29.50 （3.25）×104/ml vs 15.25 （2.25）×104/ml, P〈0.001], especially eosinophil counts [11.76 （6.02）×104/ml vs 0.12 （1.08）×104/ml, P〈0.001]. However, no significant differences of inspiratory and expiratory airway resistance and lung compliance existed in the two groups. Inspiratory and expiratory airway resistance in polymyxin B＋ fMLP group were significantly higher, lung compliance significantly lower than those in the other three groups （all P〈0.001）, and the EPX, ECP, ECF levels in serum, BALF and lung tissue of the polymyxin B＋ fMLP group were significantly higher than the other three groups （all P〈0.001）. There were more inflammatory cells, especially eosinophils in lung tissue of polymyxin B and polymyxin B＋ fMLP group. Meanwhile, more activated eosinophils in polymyxin B＋ fMLP group were observed by electron microscopy.ConclusionA mouse model of eosinophilic bronchitis can be successfully developed by transnasal administration of polymyxin B, and the eosinophil activation plays an important role in the occurrence of airway hyperresponsiveness.