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目的探讨螨变应原Der p2经聚乳酸-羟基乙酸共聚物(PLGA)包载合成的纳米疫苗特异性免疫治疗小鼠过敏性鼻炎的疗效及机制。方法取雄性SPF级BALB/c小鼠30只,4~6周龄。随机分为5组,即正常组、模型组、空白PLGA治疗组、螨抗原纳米疫苗治疗组(PLGA-DP2治疗组)及螨重组蛋白Der p2治疗组,每组6只。按常规方法建立尘螨过敏性鼻炎小鼠动物模型。建模成功后,空白PLGA治疗组、PLGA-DP2治疗组和螨重组蛋白Der p2治疗组分别使用空PLGA、PLGA-Der p2纳米疫苗(含50μg Der p2蛋白)和50μg螨重组蛋白Der p2经小鼠腹部皮下注射进行免疫治疗,每隔3 d注射1次,共治疗5次。治疗结束后,取螨变应原粗浸液200μg分别滴鼻激发小鼠,激发结束后处死小鼠。在小鼠激发后的30 min内,观察并记录各组小鼠鼻部症状(喷嚏次数、搔鼻及鼻分泌物量)评分情况;检测血清中过敏原特异性抗体(Ig E)及细胞因子[白细胞介素(IL)-2、IL-4、IL-10和γ干扰素(INF-γ)]水平;观察鼻黏膜组织形态学变化。结果螨抗原纳米疫苗PLGA-DP2载药量13.6%,包封率88.4%;纳米粒直径为200~400 nm。治疗后小鼠鼻部症状评分,PLGA-DP2治疗组鼻痒(0.67±0.52)分,喷嚏(1.00±0.63)分,清涕(1.67±0.41)分;螨重组蛋白Der p2治疗组分别为(0.83±0.41)分、(1.50±0.55)分、(0.83±0.75)分。明显低于模型组,且PLGA-DP2治疗组降低更明显(P<0.05)。PLGA-DP2治疗组和螨重组蛋白Der p2治疗组与模型组比较,血清特异性Ig E及IL-4水平方面均明显降低,PLGADP2治疗组降低更显著(P<0.05);而IL-2、IL-10和INF-γ水平显著增加,PLGA-DP2治疗组增加更显著(P<0.05)。PLGA-DP2治疗组小鼠鼻黏膜病理表现,上皮细胞基本排列整齐,组织结构清晰,相对模型组黏膜下层嗜酸性粒细胞、中性粒细胞等炎性细胞减少,鼻黏膜纤毛结构相对比较完整,黏膜基层肿大程度减轻;基本与正常组一致。结论尘螨变应原纳米疫苗加以PLGA为佐剂可有效地抑制小鼠过敏性鼻炎的炎症反应,极大地提高了过敏原疫苗的疗效。
Objective To investigate the therapeutic effect and mechanism of mite allergen Der p2 immunotherapy on allergic rhinitis in mice induced by polylactic acid-glycolic acid copolymer (PLGA). Methods Thirty male SPF BALB / c mice were aged from 4 to 6 weeks old. The mice were randomly divided into 5 groups: normal group, model group, blank PLGA treatment group, mite antigen nano vaccine treatment group (PLGA-DP2 treatment group) and mite recombinant protein Der p2 treatment group, 6 rats in each group. According to the conventional method, an animal model of dust mite allergic rhinitis was established. After successful modeling, empty PLGA, PLGA-Der p2 nano vaccine (containing 50 μg Der p2 protein) and 50 μg mite recombinant protein Der p2 were treated with empty PLGA, PLGA-DP2 and Der p2 treated groups respectively Subcutaneous injection of mice for immunotherapy, every 3 d injection 1, a total of 5 treatment. After the treatment, 200μg of crude extract of mite allergen was taken to excite the mice intranasally, and the mice were killed after the stimulation. Within 30 minutes after the mice were challenged, nasal symptom scores (number of sneezing, nasal and nasal discharge) were observed and recorded. Serum allergen-specific antibodies (Ig E) and cytokines (IL-2, IL-4, IL-10 and IFN-γ) in the nasal mucosa of the nasal mucosa. The morphological changes of nasal mucosa were observed. Results The mite antigen nanoparticle PLGA-DP2 drug loading was 13.6% and encapsulation efficiency was 88.4%. The diameter of nanoparticle was 200-400 nm. The scores of nasal symptoms, the nasal itching (0.67 ± 0.52), sneezing (1.00 ± 0.63), and clear nasal discharge (1.67 ± 0.41) in PLGA-DP2 treatment group were significantly higher than those in the control group 0.83 ± 0.41) points, (1.50 ± 0.55) points, (0.83 ± 0.75) points. Significantly lower than the model group, and PLGA-DP2 treatment group decreased more significantly (P <0.05). Compared with model group, the levels of serum specific IgE and IL-4 in PLGA-DP2 treatment group and mite recombinant protein Der p2 treatment group were significantly decreased (P <0.05), while those in PLGADP2 treatment group were significantly lower (P <0.05) The level of IL-10 and INF-γ increased significantly, while PLGA-DP2 increased more significantly (P <0.05). The histopathology of nasal mucosa in mice treated with PLGA-DP2 showed that the epithelial cells were arranged neatly and the histological structure was clear. Compared with the model group, the number of inflammatory cells such as eosinophils and neutrophils in the submucosa decreased, the structure of nasal mucosa was relatively intact, Mucosal swelling of the basement to reduce; basic and normal group. Conclusion Dust mite allergen vaccine with PLGA adjuvant can effectively inhibit the allergic rhinitis in mice and greatly improve the efficacy of allergen vaccine.