Objective To investigate the effects of down-regulating of Rfng gene ( 1, 3-Nacetylglucosaminyltransferases) in lung CD4 + T cells of asthmatic rat model by small interfering RNA ( siRNA) and explore the role of Rfng in pathogenesis of asthma. Methods An asthmatic rat model was established by OVA sensitization and challenge. Total T cells were isolated from lung tissue of asthmatic rats, and CD4 + T lymphocytes were purified using magnetic beads. CD4 + T lymphocytes were transfected by siRNA targeting Rfng gene. The mRNA and protein expressions of Rfng were detected by quantitative PCR and Western blot. Quantitative PCR was performed to determine the mRNA levels of Th1 /Th2 cytokines and related genes including IL-12, IFN-γ, IL-4, IL-5, T-bet, and GATA3. ELISA was performed to determine the concentrations of IL-12, IFN-γ, IL-4, and IL-5 in supernatant. Results The mRNA and protein expression of Rfng in RNAi group decreased significantly. IL-12, IFN-γ, T-bet increased and while IL-4, IL-5, and GATA3 decreased significantly. The concentrations of IL-12 and IFN-γ in the supernatant increased significantly, while IL-4 and IL-5 decreased significantly. Conclusions Down regulation of Rfng affects T cell differentiation. It is presumed that Fringe contribute to the pathogenesis of asthma.
Objective To investigate the expression of the histone deacetylases 1( HDAC1) and the level of whole histone acetylation and methylation in lung T cells of asthmatic rats, and investigate their role in the pathogenesis of asthma.Methods Sixteen wistar rats were randomly divided into a control group and an asthma group( n =8 in each group) . The rats was sensitized with ovalbumin( OVA) and challenged with aerosol OVA to establish asthma model. The asthmatic ratmodel was confirmed by measurement of pulmonary function, histochemical staining, HE staining, and the levels of interleukin-4 ( IL-4 ) , interferon-gamma ( IFN-γ) and immunoglobulin E( IgE) in serum and bronchoalveolar lavage fluid ( BALF) . T cells were isolated fromrat lungs and the purity was identified. The expression of the HDAC1, the level of whole histone H3 and H4 acetylation, and whole H3K9 dimethylation were analyzed by Western blot in lung T cells. Results Compared with the control group, the protein expression of HDAC1 was significantly lower( 0. 465±0. 087 vs 0. 790 ±0. 076, P lt;0. 05) in lung T cells of the asthma group. No significant differences werefound in regard to the level of whole histone H3 and H4 acetylation and whole H3K9 dimethylation betweenthe two groups. Conclusions HDAC1 in lung T cells may be involved in the pathogenesis of asthma.Histone modification by HDAC1 may be a specific eventwith gene transcription which may not be associated with asthma.
Objective To examine the expression of promoter CpG island methylation of Notch1 gene and explore the variable sites for DNA methylation in lung CD4 + T cells of asthmatic rat models.Methods An ovalbumin ( OVA) sensitized- challenged asthmatic rat model was established. Total T cells were isolated and CD4 + T lymphocytes were purified using magnetic beads. Twenty Wistar rats were randomly divided into a control group and an asthma group ( n = 10 in each group) . CD4 + T cells were isolated by immunomagnetic beads and identified by flow cytometry ( FCM) . Realtime PCR was employed to examine the mRNA expression of Notch1 gene in lung CD4 + T cells and the methylation level of Notch1 gene was examined by methylation-specific PCR. Results The mRNA expression of Notch1 in lung CD4 + T cells of the asthma group was 2. 254 ±0. 403 times as much as that of the control group. The total methylation level of asthma group was lower than that of the control group ( 0. 150 ±0. 108 vs. 0. 300 ±0. 667, P lt;0. 01) . Conclusion Promoter demethylation is one of the major mechanisms of Notch1 gene up-regulation in pathogenesis of asthma.