ABSTRACT
AIM: To investigate the effect of T-bet plasmid gene transfer to airway on allergen induced airway inflammation in a murine asthmatic model. METHODS: A mouse asthma model was established by sensitization with ovalbumin (OVA). Forty C57BL/6 mice were divided into 4 groups (10 mice in each group): the normal control group (group A), the asthmatic model group (group B), the pcDNA3 plasmid group (group C), and the pcDNA3-T-bet group (group D). The animals in group B, C and D were sensitized and challenged with OVA. The animals in group A were applied with normal saline. pcDNA3 plasmid at dose of 50 μg was intranasally administered at 24 h before intranasal challenges to the mice in group C, and the 50 μg pcDNA3-T-bet plasmid for the mice in group D. Bronchial alveolar lavage fluid (BALF) was collected and lung tissues were resected at 48 h after OVA challenge for later assay. RESULTS: After administration with pcDNA3-T-bet plasmid, high level of T-bet expression at 48 h was detected in the lung tissue by Western blotting. In pcDNA3-T-bet treated asthmatic models, histological evaluation revealed the significant suppression of eosinophil peribronchial and perivascular infiltration, and reduction of epithelial damage. The numbers of eosinophils, neutrophils and lymphocytes in BALF from pcDNA3-T-bet treated mice were significantly reduced compared to those in asthmatic control group (P<0.05). The level of IL-4 in BALF was significantly decreased in pcDNA3-T-bet group compared to that in asthmatic control group (P<0.05), while the level of IFN-γ in BALF was significantly increased in pcDNA3-T-bet group. No significant change of inflammation cells and cytokines in pcDNA3 plasmid group and asthmatic control group was observed (P>0.05). CONCLUSION: Intranasal pcDNA3-T-bet plasmid transfer inhibits asthmatic airway inflammation in the murine asthmatic model, suggesting a new therapeutic strategy for allergic asthma.
ABSTRACT
AIM: To construct recombinant adenovirus containing transcription factor T-bet (T-box expressed in T cells),and induce type-1 T-helper differentiation of lymphocytes. METHODS: T-bet gene was cloned from total RNA of lymphocyte stimulated with IFN-? with RT-PCR methods,then subcloned into transfer vector pAdtrack-CMV in BgIII/SaII sites. The new transfer vector pAdtrack-CMV. T-bet was digested with Pme I,subsequently cotransformed into BJ5183 cells with adenoviral backbone plasmid pAdEasy-1. The resultant plasmid pAd. T-bet was linearized by Pac I and transfected into 293 cells with liposome LIPOFECTAMINE 2000 for producing Ad.T-bet. The recombined adenovirus Ad.T-bet was identified through RT-PCR and Western blotting methods. Lymphocytes purified from patients suffering from liver cancer was infected with liposome and Ad.T-bet with multiplicity of infection (m.o.i) 5000,and the concentration of IFN-? in culture media was evaluated with ELISA methods. RESULTS: T-bet gene was successfully cloned from lymphocytes and incorporated into recombinant adenovirus Ad.T-bet. Lymphocytes infected with Ad. T-bet constantly and strongly secreted Th1 cytokine IFN-?. CONCLUSION: Recombinant adenovirus Ad.T-bet effectively induces type-1 T-helper differentiation,which is a promising method for restoration of patients' immune reaction against cancer.