构建以中性粒细胞浸润为主的小鼠重症支气管哮喘模型的初步探讨

目的：探讨构建以中性粒细胞浸润为主的重症支气管哮喘(简称哮喘)小鼠模型的方法。方法 C57BL/6小鼠15只,随机分成 NS 组(生理盐水组)、第1 组(50μg HDM+50μg OVA +15μg LPS)、第2 组(50μg HDM+100μg OVA+15μg LPS)、第 3组(100μg HDM+50μg OVA+15μg LPS)、第4组(100μg HDM+100μg OVA+15μg LPS),每组3只。于实验第0、1、2天腹腔注射致敏,第14、15、18、19天进行激发,第21天处理小鼠,检测相关指标。结果①小鼠行为学变化：致敏后第1~4组小鼠出现不同程度挠耳抓鼻、躁动不安、呼吸加快、大小便失禁等改变,第4组甚至出现嗜睡、匍匐不起等症状,而 NS 组未见上述表现。②小鼠肺功能：与 NS 组、第1 ~3组比较,第4组 小 鼠 在 乙 酰 甲 胆 碱 (Mch)刺激下气道阻力明显升高,呈现明显气道高反应性(P <0.05)；NS、第1、第2、第 3组小鼠气道阻力变化不明显。③小鼠肺泡灌洗液细胞总数及分类计数：与 NS 组、第1 ~3组比较,第4组小鼠 BALF 中细胞总数最多,相较 NS 组有3倍以上升高(F =66.971,P <0.001)；且第4组小鼠 BALF 中性粒细胞数量最高,约3×105/ml；与 NS 组、第1 组比较,第4组小鼠 BALF 嗜酸粒细胞数量偏高(F =23.888,P <0.01),而第2-4组比较,3组 BALF嗜酸粒细胞数量差异无统计学意义(P >0.05)。④小鼠肺组织病理：HE 染色,NS 组小鼠可见各级支气管及肺泡结构正常,支气管黏膜上皮完整连续,纤毛排列整齐,未见明显炎性细胞浸润；第1 ~4组小鼠可见不同程度支气管黏膜充血水肿明显、管腔变狭窄,杯状细胞增生及上皮细胞的坏死脱落,而气道、肺间质和小血管周围散布着大量的炎性细胞,而 第4组 小 鼠 肺 部 炎 症 浸 润 最 明 显(χ2=23.888,P <0.05)；粒细胞分化抗原-1免疫组织化学染色,NS 组小鼠未见明显中性粒细胞浸润,第1~4组小鼠可见不同程度中性粒细胞浸润,而第4组小鼠肺部中性粒细胞浸润最明显(F =28.411, P <0.05)；嗜酸粒细胞阳离子蛋白免疫组织化学染色,NS 组小鼠未见明显嗜酸粒细胞浸润,第1 ~4组小鼠可见不同程度嗜酸粒细胞浸润,但差异无统计学意义(F =0.206,P >0.05)；中性粒细胞/嗜酸粒细胞比值比较,第4组 小 鼠 肺 组 织 比 值 最 高。结 论 在 15μg LPS 环境下,100μg HDM 联合100μg OVA 可成功建立以中性粒细胞浸润为主的小鼠重症哮喘模型。

Discussion of establishing a murine model for severe asthma with the feature of neutrophil-predominate ;infiltration

Objective To explore the project of establishing a murine model for severe asthma with the feature of neutrophil-predominate infiltration.Methods Fifteen C57BL/6 mice were randomly divided into five groups:NS group (saline),group 1 (50 μg HDM+ 50 μg OVA+ 1 5 μg LPS),group 2 (50 μg HDM+100 μg OVA+1 5 μg LPS),group 3 (100 μg HDM+50 μg OVA+1 5 μg LPS),group 4 (100 μg HDM+100 μg OVA+1 5 μg LPS).Mice were administered with intraperitoneal antigen on days group 1 and 2,and challenged with OVA atomization and HDM intranasal on days 14,1 5,18 and 1 9,on 21st day the mice were killed and detected related indicators to evaluate model.Results ①Behavioral changes of mice:groups 1 to 4 after sensitization act with varying degrees of nose caught and ear scratching, irritability,rapid breathing,incontinence and other changes,even group 4 afford drowsiness and creeping, but no above performance in NS group.②Lung function results of mice:compared with NS,groups 1 to 3,group 4 mice in methacholine(Mch) stimulation significantly increased in airway resistance,showing a significant airway hyper-responsiveness(P <0.05);whereas NS,groups 1 to 3 mice airway resistance did not change significantly.③Total and differential cell count results in BALF:compared with NS,groups 1, 2,3,groups 4 total cell numbers in BALF were the highest and even more than three times NS group (F =66.971,P <0.001);with NS,group 1,2,3,the numbers of neutrophils from BALF in group 4 were the highest,approximately 3 × 10 5/ml;with NS,group 1,group 4 in BALF eosinophils infiltration was significantly(F =23.888,P <0.01),while the eosinophils infiltration from BALF in group 2,3,4 was no significant difference(P > 0.05).④ Lung pathology findings:HE stain,NS group were seen at all levels of the bronchial and alveolar structure normal bronchial continuous epithelial integrity,cilia arranged in neat rows,no significant inflammatory cell infiltration.Group 1 to 4 were seen in varying degrees of bronchial mucosal edema,stenosis,goblet cell hyperplasia and epithelial cell necrosis,airway,interstitial tissue of pulmonary and small blood vessels were around a large number of inflammatory cells,and group 4 pulmonary inflammatory infiltration significantly(χ2 =48.21 5,P <0.05).Gr-1 Immunohistochemical stain,NS group were no significant infiltration of neutrophils,group 1 to 4 mice seen varying degrees of neutrophil infiltration,and groups 4 of lung neutrophil infiltration was significantly (F = 28.41 1,P <0.05).ECP Immunohistochemical stain,NS group were no significant infiltration of eosinophil,compared with the NS group,group 1 to 4 seen varying degrees of eosinophil infiltration,while lung eosinophil infiltration was no significant difference (F = 0.206,P > 0.05).Compared with NS,groups 1 to 3, neutrophil/eosinophil ratio in group 4 was the highest.Conclusions In the presence of 1 5 μg of LPS environment,100 μgHDM + 100 μgOVA can a mice model for severe asthma with the feature of neutrophil-predominate infiltration.