In Vitro Regulation of Th1/Th2 Cell of PBMCs in AR Mice by si-GATA-3

Yuxiao LI; Ming HE; Tianrong WANG; Yonggang YAO; Yu WANG

doi:10.12259/j.issn.2095-610X.S20220802

Volume 43 Issue 8

Jul. 2022

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Yuxiao LI, Ming HE, Tianrong WANG, Yonggang YAO, Yu WANG. In Vitro Regulation of Th1/Th2 Cell of PBMCs in AR Mice by si-GATA-3[J]. Journal of Kunming Medical University, 2022, 43(8): 7-16. doi: 10.12259/j.issn.2095-610X.S20220802

Citation:

Yuxiao LI, Ming HE, Tianrong WANG, Yonggang YAO, Yu WANG. In Vitro Regulation of Th1/Th2 Cell of PBMCs in AR Mice by si-GATA-3[J]. Journal of Kunming Medical University, 2022, 43(8): 7-16. doi: 10.12259/j.issn.2095-610X.S20220802

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In Vitro Regulation of Th1/Th2 Cell of PBMCs in AR Mice by si-GATA-3

doi: 10.12259/j.issn.2095-610X.S20220802

1.
Dept. of Otorhinolaryngology-Head and Neck Surgery，The 1st Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650032
2.
Dept. of Biochemistry and Molecular Biology，School of Basic Medicine，Kunming Medical University，Kunming Yunnan 650500
3.
Key Laboratory of Animal Models and Human Disease Mechanisms，Kumming Institute of Zoology，Chinese Academy of Sciences，Kunming Yunnan 650223
4.
Dept. of Otorhinolaryngology，The Seven Affiliated Hospital，Sun-Yat Sen University，Shenzhen Guangdong 517108，China

Received Date: 2022-03-02
Publish Date: 2022-08-25

Abstract

Abstract

Objective To investigate regulatory effect of the Lentivirus-mediated-GATA-3 RNAi （si-GATA-3）on the Th1/Th2 imbalance in a murine model of allergic rhinitis in vitro. Methods The lentiviral vector SI-GATA-3 with GATA-3 gene mediated by RNAi was developed and packaged, purified and titrated. BALB/C mice were randomly divided into AR group and normal control group. The allergic rhinitis model of BALB/C mice was developed by using ovalbumin （OVA）. The peripheral blood of BALB/C mice was collected and their mononuclear cells （PBMCs） were isolated. The PBMCs were randomly divided into three groups, namely, si-GATA-3 group, si-NC group and AR group. si-GATA-3, si-NC and normal saline were used to intervene PBMCs in allergic rhinitis mice for 72 h, and normal saline was used to replace si-GATA-3 to intervene PBMCs in normal control mice. PBMCs and cell culture supernatant were separated after the intervention. The relative expression levels of GATA-3 mRNA, T-bet mRNA and GATA-3, T-bet protein in PBMCs were detected by fluorescence quantitative qPCR and Western bloting, respectively. The contents of IL-4 and IFN-γ in supernatant of cell culture were determined by ELISA. Results The RNAi mediated lentiviral vector si-GATA-3 with a titer of 5×10⁸ TU/mL was successfully deeloped. The allergic rhinitis mice models were established successfully. The relative expression levels of GATA-3 mRNA and GATA-3 protein and the content of IL-4 in supernatant of PBMCs in si-GATA-3 group were significantly lower than those in AR group and si-NC group （P < 0.01）, and the expression levels of GATA-3 in AR group and si-NC group were significantly higher than those in normal control group （ P < 0.01）. There was no difference between AR group and si-NC group （ P > 0.05）. The relative expression of T-bet mRNA and T-bet protein in si-GATA-3 group was significantly higher than that in AR group and si-NC group （ P < 0.01）, and the expression of T-bet mRNA and T-bet protein in AR group and si-NC group was significantly lower than that in control group （ P < 0.01）. There was no difference between AR group and si-NC group （ P > 0.05）. However, the content of IFN-γ in cell culture supernatant of si-GATA-3 group was higher than that of normal control group （ P < 0.05）, and the content of IFN-γ in AR and si-NC groups was significantly higher than that of normal control group and si-GATA-3 group （ P < 0.01）. There was no significant difference in IFN-γ expression between AR group and si-NC group （ P > 0.05）. Conclusions si-GATA-3 could down-regulate the relative expression of GATA-3 mRNA , GATA-3 protein and IL-4 level in Th2 cells, and up-regulate the relative expression of T-bet mRNA and T-bet protein in Th1 cells. Therefor, it can effectively correct the immune imbalance of Th2/Th1 in PBMCs in mouse AR model.
- Rhinitis,
- Allergic,
- RNA interference,
- GATA-3,
- T-bet,
- Lentiviral vector,
- Model,
- Mouse

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