目的：制备粉尘螨粗抗原建立BALB/c小鼠哮喘模型，检测细胞因子并观察肥大细胞形态及脱颗粒的情况。方法：制备粉尘螨粗抗原，30只BALB/c小鼠随机均分为3组，阴性对照组（A组）、哮喘模型组（B组）和治疗组（C组）。A组始终使用PBS处理，B组和C组在第0、7和14天用50μg粉尘螨粗抗原＋50μl明矾佐剂腹腔注射致敏。在第28天，A组和B组用PBS、C组用粉尘螨粗抗原（350μg/次）皮下注射免疫1周，隔天免疫1次，末次免疫1周后开始用50μg粉尘螨粗抗原滴鼻激发，每天1次，连续7次。末次激发24h后进行气道高反应检测。末次激发48h后处死小鼠取血，对小鼠进行肺泡灌洗收集肺泡灌洗液（BALF），无菌摘取肺组织和脾脏，分别进行肺组织病理切片与脾细胞培养。检测BALF和脾细胞上清中IL-4、IL-10和IFN-γ细胞因子水平，血清中IgE、组胺抗体水平，进行HE染色和甲苯胺蓝染色观察肺部炎症细胞浸润程度和肥大细胞脱颗粒状况。结果：与B组相比，C组气道高反应性和肺部病理症状减轻（P＜0．01）。C组BALF中细胞计数结果显示与B组相比细胞总数和嗜酸粒细胞数明显减少（P＜0．01）。与B组相比，C组的肥大细胞脱颗粒情况明显减轻，细胞形态稳定。与B组（IgE：1．905）相比，C组血清中抗原特异性IgE抗体水平（IgE：1．278）明显降低（P＜0．01）。BALF细胞因子IL-4、IL-10和IFN-γ水平检测结果显示，与B组相比，C组BALF中IL-4水平明显降低接近A组值（P＜0．01），而IL-10与A、B两组相比有明显升高（P＜0．01），C组的IFN-γ也高于A、B两组（P＜0．01）。C组脾细胞上清中IL-4明显低于B组（P＜0．01），而IL-10明显高于B组（P＜0．01），IFN-γ也明显高于B组（P＜0．01）。BALF和血清中组胺水平结果显示，在BALF中，C组与B组相比略低（P＜0．05），而在血清中检测出的组胺有明显降低（P＜0．05）。结论：过敏性哮喘模型脱敏治疗后小鼠肥大细胞脱颗粒情况被抑制。%Objective:To prepare Dermatophagoides farinae (Der f) crude protein to establish BALB/c bronchial asthma model , and to observe the morphology and degranulation of mast cells and detect related cytokines .Methods: Dermatophagoides farinae ( Der f) crude protein were prepared by trituration .30 BALB/c mice were randomly divided into 3 groups:PBS control group (A), asthma model group (B) and Der f crude protein treatment group (C).Group A were treated with PBS(100 μl) all the time, group B and group C were treated with 50 μg Der f crude protein mixed with 50μl alum adjuvant on day 0,day 7 and day 14.On day 28 group A and B were subcutaneous injected with PBS (100 μl) and group C were subcutaneous injected with Der f crude protein (350μg) in PBS(100 μl) at 1-day intervals.One week after the last treatment ,group A,B and C were intranasally challenged with 50 μg Der f crude protein daily for seven days .Twenty-four hours after the last challenge , airway hyper-responsiveness ( AHR) was assessed by using whole-body plethysmography .Two days post challenged , mice were sacrificed and bronchoalveolar lavage fluid ( BALF) was collected.Number of the total cells and eosinophil was determined .Level of IL-4,IL-10 and IFN-γcytokines in the BALF and the su-pernatant of splenocyte culture was assayed by ELISA .Level of Der f specific IgE and histamine in the sera was determined by ELISA . Airway inflammation was analyzed by HE staining .Observation of the morphology and degranulation of mast cells was analyzed by tolui -dine blue staining.Results:Compared with group B,AHR and the lung inflammation in group C were greatly reduced (P<0.01). Numbers of total cells and eosinophils in BALF of group C were significantly lower than that of group B ( P<0.01 ) .Compared with group B, the observation of degranulation of mast cells was insignificant in group C .Compared with group B(IgE:1.905), the level of specific IgE was significantly lower in groups C (IgE:1.278)(P<0.01).The level of IL-4 in BALF of group C was significantly lower than that of group B(P<0.01).Compared with group A and B, the level of IL-10 in BALF was significantly higher in group C (P<0.01) and the level of IFN-γin BALF of group C was significantly higher than that of group A and B (P<0.01).Compared with group B, the level of IL-4 in cultured splenocytes was significantly lower in group C (P<0.01), and the level of IL-10 and IFN-γin cultured splenocytes of group C was significantly higher than that of group B (P<0.01).Compared with group B, the level of histamine in BALF was slightly lower in groups C (P<0.05), and the level of histamine in sera was significantly lower in groups C (P<0.05). Conclusion:The degranulation of mast cells of murine bronchial asthma model was suppressed after the desensitization therapy .
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