关键词: 粉尘螨, 壳聚糖, 纳米颗粒, 舌下含服疫苗, 免疫治疗

Abstract: Objective   To prepare Dermatophagoides farinae （Der f）/chitosan nanoparticle vaccine （DCN）, and to investigate the effect of sublingual administration with DCN in asthma mice model.  Methods   DCN were prepared by ionotropic gelation. 30 BALB/c mice were randomly divided into 5 groups： normal control group（A）, PBS control group （B）, Chitosan group （C）, Der f group （D）, DCN group （E）. Group A were treated with normal saline （100 μl） all the time. Mice in other groups were sensitized intraperitoneally with 50 μg dust mite extracts plus 2 mg Al（OH）3, and on day 28 given a sublingual vaccination of PBS（group B）, or empty CS nanoparticles （group C）, or Der f （group D, 1 mg Der f） or DCN （group E, loaded with 1 mg Der f）. All the mice received 18 doses at 1-day intervals. One week after the last immunization, mice in group B, C, D， and E were intranasally challenged with 50 μg Der f extract daily for seven days. Twenty-four hours after the last challenge, airway hyper-responsiveness （AHR） was assessed by using whole-body plethymography. Two days post challenge, mice were sacrificed and bronchoalveolar lavage fluid （BALF） was collected. Number of the total cells and eosinophils was determined. Level of cytokines in the supernatant of splenocyte culture was assayed by ELISA. Level of Der f specific IgE, IgG2a and IgA in the sera was determined by ELISA. Airway inflammation was analyzed by HE staining. Spleen lymphocyte proliferation responses were analyzed by MTT colorimetry.   Results   Compared with group B， AHR and the lung inflammation in groups D and E were greatly reduced. Numbers of total cells and eosinophils in BALF of groups D （36.50×10⁴/ml， 3.72×10⁴/ml） and E （34.25×10⁴/ml， 2.25×10⁴/ml） were significantly lower than that of group B （61.67×10⁴/ml， 14.17×10⁴/ml） （P＜0.05）. The level of specific IgE was significantly lower in groups D （0.22） and E （0.22）， and that of IgA in groups D （0.88） and E （1.03） was significantly higher than that in group B （0.79）. The level of IL-4 in BALF （D: 28.49 pg/ml， E: 20.93 pg/ml） and cultured splenocytes （D: 27.82 pg/ml， E: 20.80 pg/ml） of groups D and E was significantly lower than that of group B （56.33 pg/ml， 45.84 pg/ml） （P＜0.05）. While IFN-γ （D: 18.80 pg/ml， E: 37.32 pg/ml） and IL-10 （D: 118.90 pg/ml， E: 129.15 pg/ml） in BALF in groups D and E were significantly higher than that of group B （13.60 pg/ml， 29.61 pg/ml） （P＜0.05）， and same with IFN-γ （D: 20.68 pg/ml， E: 42.42 pg/ml） and IL-10 （D: 36.31 pg/ml， E: 161.37 pg/ml） in spleen cultured supernatants of groups D and E （P＜0.05）. The allergen-specific splenocyte proliferation was inhibited in groups D （SI： 0.14） and E （SI： 0.13）， and there was no significant difference between group C （SI： 0.22） and group B （SI： 0.23）.   Conclusion   Dermatophagoides farinae （Der f） /chitosan nanoparticle vaccine has therapeautic effect on murine asthma.

Key words: Dermatophagoides farinae, Chitosan, Nanoparticle, Sublingual vaccine, Immunotherapy