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Updated: May 12, 2026
Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation
Published on: April 13, 2017
[Small hairpin RNA silencing Tim-3 affects peripheral blood Th1 and Th17 cells differentiation in asthmatic mice]
Xiao-Xia Lu1, Jia-Li Xu, Zong-Qi Dong
1Department of Pulmonary Medicine, Wuhan Childern's Hospital, Wuhan, China. lusi74@163.com
Objective:
To investigate the effects of down-regulating Tim-3 gene in the peripheral blood mononuclear cells (PBMCs) of an asthmatic mouse model by short hairpin RNA (shRNA) and to explore the effect of Tim-3 on Th1 and Th17 cell differentiation.
Methods:
An asthmatic murine model was established by ovalbumin sensitization and challenge. PBMCs were isolated from asthmatic mice and transfected by shRNA targeting Tim-3 gene. The mRNA and protein expressions of Tim-3 were detected by quantitative PCR and Western blot. Flow cytometry analysis was performed to determine the levels of Th1 and Th17, and ELISA was performed to determine concentrations of IFN-γ, IL-4 and IL-17 in the supernatant.
Results:
Tim-3 mRNA expression in PBMCs was significantly increased in asthmatic mice. The mRNA and protein expression of Tim-3 decreased significantly in the shRNA group. Compared with the negative groups, Th1 cell levels increased and Th17 cell levels decreased significantly in the asthmatic groups after Tim-3 shRNA interference. In the Tim-3 shRNA interference groups concentrations of IFN-γ increased significantly while IL-17 decreased significantly.
Conclusions:
Specific Tim-3 shRNA effectively silences the expression of Tim-3 and change in Tim-3 expression could affect T cell differentiation.