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Effect of cytokine on differentiation to Th17 and secretion of IL-17 of mouse spleen mononuclear cells

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Author(s): Hui-li ZHENG | Hai-bin LI | Li XIAO | Wei ZHAO | Guo-sheng DU | Zhen WANG | Bing-yi SHI

Journal: Medical Journal of Chinese People's Liberation Army
ISSN 0577-7402

Volume: 36;
Issue: 4;
Start page: 372;
Date: 2011;
Original page

Keywords: cytokines; spleen; mononuclear cells; Th17; interleukin-17

ABSTRACT
Objective To investigate the effect of exogenous cytokine TGF-β,IL-6 and IL-23 on the differentiation to Th17 and secretion of IL-17 of mouse spleen mononuclear cells,and discuss their regulatory mechanism in organ transplantation immunity.Methods Mouse spleen mononuclear cells were stimulated by phorbol myristate acetate(PMA) and ionomycin(Ion),and co-cultured with different cytokines for 72 hours.The spleen mononuclear cells were assigned into 4 groups according to the cytokines with which they were co-cultured: blank group,TGF-β group,TGF-β+IL-6 group and TGF-β+IL-6+IL-23 group.The ratio of Th17 cells was analyzed using flow cytometry.Expressions of RORγt mRNA and IL-17 mRNA were analyzed using semi-RT-PCR.IL-17 protein in supernatant was assayed using ELISA.Results Th17 cells were not found in blank group and TGF-β group,nor were RORγt mRNA in both groups.There was statistical difference on the ratios of Th17 cells between TGF-β+IL-6 group and TGF-β+IL-6+IL-23 group(0.30%±0.14% vs 0.51%±0.11%,P < 0.01),and also on the expressions of RORγt mRNA between TGF-β+IL-6 group and TGF-β+IL-6+IL-23 group(0.217±0.038 vs 0.458±0.127,P < 0.01).There was no statistical difference on the expression of IL-17 mRNA between blank group and TGF-β group(0.868±0.222 vs 0.799±0.205,P > 0.05),while a difference was found between TGF-β+IL-6 group and TGF-β+IL-6+IL-23 group(1.396±0.359 vs 2.415±0.604,P < 0.01).There was no statistical difference on the expression of IL-17 protein between blank group and TGF-β group(90.04±22.56pg/ml vs 125.38±27.58pg/ml,P > 0.05),while difference was found between TGF-β+IL-6 group and TGF-β+IL-6+IL-23 group(973.32±217.33pg/ml vs 1498.46±317.27pg/ml,P < 0.01).The expressions of IL-17 mRNA and IL-17 protein were higher in TGF-β+IL-6 group and TGF-β+IL-6+IL-23 group than in blank and TGF-β groups(P < 0.01).Conclusions TGF-β+IL-6 may promote the differentiation and secretion of IL-17 in vitro.IL-23 may significantly promote the effect of TGF-β+IL-6.

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