调节性T细胞在肝再生增强因子免疫抑制机制中的作用研究

Role of regulatory T cells in immunosuppression of human augmenter of liver regeneration in vitro

目的观察人肝再生增强因子(augmenter of liver regeneration,ALR)对体外活化的单个核细胞中调节性T细胞(regulatory T cell,Treg)的增殖、分化及抑制性细胞因子TGF-β1、IL-10表达水平的影响,探讨ALR的免疫抑制机制。方法梯度离心分离正常人外周血单个核细胞(human peripheral blood monoclear cells,PBMCs),分为对照组(Control组)、ConA刺激组(ConA组)和人ALR(hALR)干预组(ConA+hALR组)。ConA组和ConA+hALR组分别给予ConA(5μg/mL)和hALR(30μg/mL)处理,对照组不处理。各组作用60 h后,2-对磺酸基苯基-3-(4,5-二甲基噻唑)-5-(3-羧甲氧基苯基)-二氢四唑嗡盐[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt,MTS]检测各组细胞增殖情况,流式细胞术检测各组CD4+CD25+FoxP3+Treg细胞比例和细胞周期变化,Real-time PCR检测各组FoxP3 mRNA的表达,ELISA检测各组上清中细胞因子TGF-β1、IL-10浓度。结果 hALR能明显抑制ConA活化的PBMCs的增殖(P<0.01);与ConA组和Control组相比,hALR能显著增加CD4+CD25+FoxP3+Treg细胞占CD4+T细胞的比例(P<0.01)和FoxP3 mRNA的表达(P<0.01),并且hALR能够解除ConA引起的Treg细胞周期G2/M期的阻滞(P<0.01)。此外,与ConA组和Control组相比,ConA+hALR组上清中TGF-β1和IL-10的浓度明显升高(P<0.01)。结论hALR可以通过诱导Treg细胞的增殖、分化以及促进TGF-β1和IL-10的产生,从而达到免疫抑制的作用。

Objective To determine the effect of human augmenter of liver regeneration （hALR） on the proliferation of regulatory T cells and expression levels of the inhibitory cytokines TGF-β1 and IL-10 in ConA-stimulated human peripheral blood mononuclear cells （PBMCs） in order to investigate the underlying immunosuppressive mechanism of hALR. Methods PBMCs were isolated from healthy volunteers by gradient centrifugation, and then divided into normal control group, ConA stimulated group and hALR interfering group. Cells in the ConA group and ConA＋hALR group were processed by 5 μg/mL ConA and 30 μg/mL hALR, and control group was not treated. After cultured in vitro for 60 h, MTS assay [3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium,inner salt] was used to determine the cellular viability. Flow cytometry （FCM） was used to detect the proportion of CD4^＋CD25^＋FoxP3^＋Treg and the mitotic cycle of Treg. FoxP3 mRNA was detected by real-time fluorescent quantitation PCR, and the levels of TGF-β1 and IL-10 in the supernatant were determined by ELISA. Results hALR obviously inhibited the proliferation of ConA-stimulated PBMCs （P〈0.01）. Compared with the ConA group and control group, hALR significantly increased the proportion of CD4^＋ T cells in CD4^＋ CD25^＋FoxP3^＋ Treg cells （P〈0.01） and expression of FoxP3 mRNA . In addition, hALR relieved the G2/M cell cycle arrest of Treg induced by ConA, and the levels of TGF-β1 and IL-10 in the supernatant of hALR interfering group, which was significantly higher than ConA group and control （P〈0.01）. Conclusion hALR induces the proliferation and differentiation of Treg, and increases the release of TGF-β1 and IL-10, and thus results in the inhibition of immune response.