谷氨酰胺对人外周血单个核细胞细胞因子表达的影响

Effects of glutamine on the cytokine expression of peripheral blood mononuclear cells

目的探讨谷氨酰胺（Gln）对内毒素（LPS）刺激下健康人外周血单个核细胞（PBMC）细胞因子表达的影响。方法取健康志愿者新鲜外周血，利用密度梯度离心法提取单个核细胞，观察内毒素刺激单个核细胞表达肿瘤坏死因子（TNF-α）和白细胞介素（IL）系列（IL-1β、IL-4、IL-6、IL-8、IL-10），酶联免疫吸附法（ELISA）检测上清中细胞因子的表达，SuperArray基因芯片技术检测细胞中细胞因子mRNA的表达，同时分别使用1、8、10和15mmol／L Gln预处理单个核细胞0．5h和2h后观察细胞因子在蛋白以及基因水平的表达。结果LPS刺激PBMC后观察部分细胞因子表达明显增强；1mmol／L Gln组细胞因子表达无变化，8mmol／L Gln预处理后TNF-α、IL-1β、IL-8和IL-10表达下降（P〈0．05），15mmol／L Gln组炎症介质表达均有显著下降（P〈0．01）。但与未受LPS刺激组相比炎症介质表达仍有增高（P〈0．05）。Gln预处理2h组细胞因子表达的抑制较0．5h明显增强（P〈0．05），并且存在时间和剂量的依赖性。结论Gln能够下调LPS刺激下PBMC细胞因子的过表达。

Objective To investigate the effects of glutamine （Gln） on the cytokine expression of the peripheral blood mononuclear cells （PBMCs） stimulated by lipopolysaccharide （LPS）. Methods PBMCs were extracted from healthy volunteer by density gradient centrifugation. Suspension of PBMCs was pretreated with Gln of the concentrations of 1 mmol/L,8 mmol/L,10 mmol/L,and 15 mmol/L for 0. 5 h and 2 h respectively. Suspension of PBMCs without Gln pretreatment but stimulated by LPS was used as positive control group, and suspension without LPS stimulation was used as negative control group. Then LPS was added ELISA was used to examine the levels of the mediators of inflammation, TNF-α, IL-1β, IL-4, IL-6, IL-8, and IL-10, in the supernatant, and Gene Array Protocol was used to examine the mRNA expression of these cytokines. Result After LPS stimulation, the expression levels of all the cytokines were obviously enhanced. The expression levels of the proinflammatory mediator, except for IL-6, of the Gln 8 mmol/L 0. 5 h group were significantly lower than those of the positive control group; and the expression levels of all the cytokines of the Gln 15 mmol/L 0. 5 h group were significantly lower than those of the positive control group, however, all still significantly higher than those of the negative control group （P 〈0.01 or P 〈0. 05）. The expression levels of the proinflammatory mediator except for IL-8 of the Gln 1 mmol/L 0. 5 h group did not significantly changed in comparison with the positive control group; and the level of IL-8 was significantly decreased （P 〈 0.01 ）. The expression levels of all the cytokines of the high concentrations Gln groups were all significantly lower than those of the positive control group. The levels of TNF-α, IL-6, and IL-8 of the high concentration Gln 2 h groups were all significantly higher then those of the concentration Gln 0. 5 h groups （ all P 〈 0. 01 ）, however, there was not significant difference in the level of IL-1β between the high concentration 0. 5 h and 2 h Gln groups. The level of the anti-inflammatory mediator IL-10 of the high concentration Gln 0. 5 h and 2 h groups, especially that of the latter group, were significantly lower than that of the positive control group （ beth P 〈 0. 01 ）, however, the IL-10 level of the high concentration Gln 0. 5 h group was still significantly higher than that of the negative control group （ P 〈 0.05 ）. Gene chip analysis confirmed the above results. Conclusion Glutamine dose- and time-dependently downregulates the expression of the mediators of inflammation, and lessen the excessive expression of those cytokines in cells stimulated by LPS.