5-氮杂胞苷对T淋巴细胞Jurkat miR-126及IFN-γ、GATA3、ROR-γ、Foxp3表达的影响

Effects of 5-azacytidine on Expression of Jurkat miR-126 and IFN-γ,GATA3, ROR-γ and Foxp3 in T Lymphocytes

目的:探究甲基化酶抑制剂5-氮杂胞苷(5-azacytidin,5-aza)对T淋巴细胞(Jurkat)mi R-126、Th1/Th2、Th17/Treg细胞亚群及因子IFN-γ、GATA3、ROR-γ和Foxp3的调控作用。方法:采用不同浓度5-aza干预T淋巴细胞,24 h、48 h后检测其对细胞增殖抑制作用;实时荧光定量PCR、Western Blot检测5-aza干预后mi R-126表达水平以及IFN-γ、GATA3、ROR-γ和Foxp3的m RNA及蛋白表达水平;流式细胞术检测5-aza干预后Th1/Th2、Th17/Treg细胞亚群分化比例。结果:甲基化酶抑制剂干预T淋巴细胞后,细胞抑制率随5-aza浓度增大及作用时间延长呈递增趋势(P<0.01);细胞抑制率在24 h、48 h,低、中、高浓度下分别为(14.73±0.93)%、(32.67±8.40)%、(60.87±5.78)%以及(18.98±0.73)%、(39.80±8.42)%、(64.11±6.04)%;抑制率在24 h与48 h之间无差异(P>0.05)。甲基化酶抑制剂干预后mi R-126表达降低(P<0.01);IFN-γ蛋白表达降低(P<0.05)、Th1细胞亚群数目降低(P<0.01);GATA3 m RNA和蛋白表达升高(P<0.05)、Th2细胞亚群数目增加(P<0.01);ROR-γ蛋白表达降低(P>0.05)、Th17细胞亚群数目降低(P<0.05);Foxp3 m RNA和蛋白表达升高(P>0.05)、Treg细胞亚群数目增加(P<0.05)。结论:甲基化酶抑制剂可以下调Jurkat细胞mi R-126基因表达;下调Th1、Th17细胞亚群分化,上调Th2、Treg细胞亚群分化;调控细胞因子IFN-γ、GATA3、ROR-γ和Foxp3的表达。

Objective: To investigate the regulatory effects of methylase inhibitor 5-azacytidin(5-aza) on mi R-126, Th1/Th2,Th17/Treg cell subsets and cytokines IFN-γ, GATA3, ROR-γ and Foxp3 in T lymphocytes(Jurkat). Methods: T lymphocytes were treated with different concentrations of 5-aza. The inhibitory effects of 5-aza on T lymphocytes proliferation were measured at 24 h and48 h after treatment. Real-time quantitative PCR and Western Blot were used to detect the expression level of mi R-126 and the m RNA and protein expression levels of IFN-γ, GATA3, ROR-γ and Foxp3 after 5-aza intervention;Flow cytometry was used to detect the differentiation ratio of Th1/Th2 and Th17/Treg cell subsets after 5-aza treatment. Results: The cytostatic rate increased with increasing5-aza concentration and duration of action after methylation enzyme inhibitor intervention on T lymphocytes(P<0.01). The cell inhibitory rates were(14.73±0.93)%,(32.67±8.40)%,(60.87±5.78)% and(18.98±0.73)%,(39.80±8.42)%,(64.11±6.04)% at 24 h, 48 h, low,medium and high concentrations, respectively. There was no difference in inhibition between 24 h and 48 h(P>0.05). The expression of mi R-126 decreased after methylase inhibitor intervention(P<0.01);The expression of IFN-γ protein decreased(P<0.05), the number of Th1 cell subsets decreased(P<0.01);GATA3 m RNA and protein expression increased(P<0.05), the number of Th2 cell subsets increased(P<0.01);The expression of ROR-γ protein was decreased(P>0.05), the number of Th17 cell subsets decreased(P<0.05);Foxp3 m RNA and protein expression increased(P>0.05), the number of Treg cell subsets increased(P<0.05). Conclusions: Methylase inhibitors could down-regulate mi R-126 gene expression in Jurkat cells. The down-regulation of subpopulation differentiation of Th1 and Th17 cells, up-regulation of subpopulation differentiation of Th2 and Treg cells, and expression of cytokines IFN-γ, GATA3, ROR-γ and Foxp3 were regulated by methylase inhibitors.