ABSTRACT
Objective To construct the adeno-associated virus (AAV)vector of mouse carrying T-bet gene,which was applied to mouse model of asthma by nose,and to investigate the immunomodulatory effects of T-bet delivery on asthma.Methods Forty healthy Balb/c mice (aged 6-8 weeks) were randomly divided into 4 groups:control group (group A),asthma model group(group B),model/control recombinant adeno-associated virus carrying enhanced green fluorescent protein(rAAV-eGFP) intervention group (group C) and model/rAAV-T-bet virus intervention group(group D),with 10 mice in each group.In group B,group C,and group D,Balb/c mice were sensitized by intraperitoneal injection with OVA and challenged by nebulized OVA.In group C and group D,Balb/c mice were intervened with the isodose rAAV-eGFP and rAAV-T-bet,while in group B,the mice were intervened with the same amount of saline,both of them were dropped into the nasal cavity.Twenty-four hours after the last injection,the mice were sacrificed and the samples were obtained.The total cells in bronchoalveolar lavage fluid (BALF) were counted,and the types of cells were analyzed by Wright-Giemsa staining.The levels of interferon-γ (IFN-γ),IL-4 and IL-5 in BALF were determined by enzyme-linked immunosorbent assay.The expressions of T-bet,GATA-3 and Foxp3 protein in the lungs of asthmatic mice were detected with immunohistochemical technique.Results 1.The level of specific transcription factor T-bet in group B and group C were distinctly lower than that of group A (all P ＜ 0.05),while the level of GATA-3 was significantly higher than that of group A(all P ＜ 0.05).In group D,the expression of T-bet protein was stronger than that in group B and group C,however,the expression of GATA-3 decreased obviously (all P ＜ 0.05).The results of Foxp3 were similar to T-bet.2.In group D,the mice had suppressed levels of IL-4 [(158 ± 55) ng/L] and IL-5 [(68-± 22) ng/L] compared with those in group B and group C(all P ＜0.05).The level of IFN-γ[(113-±35) ng/L] increased compared with those in group B and group C (all P ＜ 0.05).At the same time,the number of total cells and eosinophil in BALF were both obviously lower than those of group B and group C (all P ＜ 0.05).Conclusions On the basis of building mouse model of asthma,intranasal administration of rAAV-T-bet can deliver T-bet gene to airway successfully and efficiently,and plays an immunomodulatory role in immune confusion of asthma.