TNF-α与特发性血小板减少性紫癜患者激素抵抗的相关性研究

Study on the relationship of tumor necrosis factor-α and glucocorticoid-resistant idiopathic thrombocytopenia purpura

目的研究特发性血小板减少性紫癜(idiopathic thrombocytopenia purpura,ITP)患者肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的表达及其对糖皮质激素受体(glucocorticoid receptor,GR)表达的影响,探讨TNF-α与糖皮质激素抵抗的相关性。方法ELISA方法检测ITP激素敏感型、激素抵抗型患者及正常对照组的血浆中TNF-α的含量;分离健康志愿者外周血单个核细胞(PBMC),一组用TNF-α刺激培养,一组单独培养作为对照,分别采用RT-PCR方法及Western blot法分析其GRα和GRβmRNA及蛋白表达的变化。结果血浆TNF-α浓度在ITP患者激素敏感组、激素抵抗组、正常对照组分别为(13.69±4.92)pg/mL、(19.73±4.66)pg/mL、(7.51±2.79)pg/mL,二组ITP患者血浆中TNF-α的含量均高于正常对照组(P<0.01),其中激素抵抗组ITP患者血浆中TNF-α的含量高于激素敏感组(P<0.05);经TNF-α刺激培养24 h后,培养组与对照组GRα/GAPDHmRNA分别为1.16±0.73,1.20±0.51,培养组与对照组相比,其GRαmRNA表达无显著变化(P>0.05);培养组与对照组GRβ/GAPDHmRNA分别为1.62±0.59,0.81±0.66,培养组与对照组相比,GRβmRNA表达明显增加(P<0.05);GRα蛋白的表达在各组之间无显著差别,但GRβ蛋白的表达在TNF-α刺激组较对照组明显增高。结论ITP激素抵抗型患者血浆TNF-α含量增高;TNF-α可以增加PBMC中GRβ的表达,这可能是炎性细胞因子诱发糖皮质激素抵抗的内在机制。

Objective To study the expression of tumor necrosis factor-alpha （TNF-α） in idiopathic thrombocytopenia purpura （ITP） patients and the effect of TNF-α on the expression of human glucocorticoid receptor（GR） in order to explore the relationship of TNF-α and glucocorticoid resistance. Methods The plasma TNF-α concentrations were measured by enzyme-linked immunosorbent assay. （ELISA） in glucocorticoid sensitive and glucocorticoid resistant ITP patients. Human peripheral blood mononuclear cells （PBMC）from healthy volunteers were isolated and the mRNA expressions and protein expressions of GRα and GRβ in PBMC being cultured with TNF-α were detected by RT-PCR and Western blot. Results The plasma TNF-α concentrations in ITP groups were significantly higher than that in controls （P 〈 0. 01 ）. The plasma TNF-α concentrations in the glucocorticoid resistant group was significantly higher than that in the glucocorticoid sensitive group （ P 〈 0.05 ）. After being cultured with TNF-α for 24 h,the expression of hGRα mRNA in PBMC of the cultured group was identical to that of the controls （P 〉0. 05） ,which GRot/GAPDH mRNA of the cultured group and the controls were 1.16 ±0.73 and 1.20 ± 0. 51 ,respectively. But the expression of hGRβ mRNA in the cultured group was significantly higher than that in the controls （ P 〈 0.05 ）, the GRβ/GAPDH mRNAs of the cultured group and the controls were 1.62 ± 0. 59 and 0.81 ± 0.66, respectively. The GRct protein expressions were similar in both groups, but the GRβ protein expression in TNF-α- stimulated group was higher than that in the controls. Conclusion The plasma TNF-α concentrations were significantly higher in the glucocorticoid resistant ITP patients. TNF-α can increase the expression of GRβ,which may be one of the mechanisms of glucocorticoid resistance induced by inflammatory cytokines.