Abstract 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.
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［8］Agache I, Ciobanu C, Agache C, Anghel M. Increased serum IL-17 is an independent risk factor for severe asthma［J］. Respir Med, 2010, 104(8): 1131-1137.
［9］Wilson RH, Whitehead GS, Nakano H, Free ME, Kolls JK, Cook DN. Allergic sensitization through the airway primes Th17-dependent neutrophilia and airway hyperresponsiveness［J］. Am J Respir Crit Care Med, 2009, 180(8): 720-730.
［10］Nakae S, Komiyama Y, Nambu A, Sudo K, Iwase M, Homma I,et al. Antigen-Specific T Cell Sensitization Is Impaired in IL-17-Deficient Mice, Causing Suppression of Allergic Cellular and Humoral Responses［J］. Immunity, 2002, 17(3): 375-387.
［12］Nakae S, Iwakura Y, Suto H, Galli SJ. Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17［J］. J Leukocyte Biol, 2007, 81(5): 1258-1268.
［14］Kurschus FC, Croxford AL, Heinen AP, Wortge S, Lelo D, Waisman A. Genetic Proof for the transient nature of the Th17 phenotype［J］. Eur J Immunol, 2010, 40(12): 3336-3346.
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［17］Darcan-Nicolaisen Y,Meinicke H,Fels G, Hegend O, Haberland A, Kuhl A, et al. Small interfering RNA against transcription factor STAT6 inhibits allergic airway inflammation and hyperreactivity in mice［J］. Immunol, 2009, 182(12): 7501-7508.
［18］Moriwaki A, Inoue H, Nakano T, Matsunaga Y, Matsuno Y, Matsumoto T, et al. T Cell treatment with small-interfering RNA for suppressor of cytokine signaling 3 modulates allergic airway responses in a Murine Model of Asthma［J］. Am J Respir Cell Mol Biol, 2011, 44(4): 448-455.
［19］Bernards R, Brummelkamp TR, Beijersbergen RL. shRNA libraries and their use in cancer genetics［J］. Nat Methods, 2006, 3(9):701-706.
