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目的观察肿瘤坏死因子(TNF)-α对血栓调节蛋白(TM)在原代人脐静脉内皮细胞(HUVECs)的表达和活性的影响,并初步探讨其作用机制。方法体外培养HUVECs,实验分为3组:1对照组:加入与TNF-α等体积的PBS培养细胞6 h;2 TNF-α组:TNF-α终浓度为10 ng/ml孵育细胞6 h;3 BAY11-7082抑制组:BAY11-7082终浓度2μg/ml孵育细胞1 h阻断NF-κB通路后再用10 ng/ml的TNF-α干预细胞6 h。分别采用流式细胞技术、荧光定量聚合酶链反应(RT-PCR)和酶标仪检测TM蛋白、mRNA表达及活性强度。结果 TNF-α组相比与对照组和BAY11-7082抑制组在TM的蛋白、mRNA水平上均明显降低(P<0.05),TM活性也明显减弱(P<0.05)。而BAY11-7082抑制组相比与对照组在TM蛋白水平和活性均无差异,在mRNA水平上升高(P<0.05)。结论 TNF-α可明显降低TM表达,并抑制其活性,NF-κB通路参与了这一过程。
Objective To investigate the effect of tumor necrosis factor-α (TNF-α) on the expression and activity of thrombomodulin (TM) in primary human umbilical vein endothelial cells (HUVECs) and to explore its possible mechanism. Methods HUVECs were cultured in vitro. The experiment was divided into three groups: 1 control group: 6 hours incubation with equal volume of TNF-α; 2 TNF-α group: 6 hours incubation with 10 ng / ml TNF- 3 BAY11-7082 inhibition group: BAY11-7082 cells were incubated with 2μg / ml BAY11-7082 for 1 h, then blocked with 10 ng / ml TNF-α for 6 h after blocking NF-κB pathway. Flow cytometry, fluorescence quantitative polymerase chain reaction (RT-PCR) and microplate reader were used to detect TM protein, mRNA expression and activity intensity respectively. Results Compared with the control group and the BAY11-7082 inhibition group, the levels of TM protein and mRNA in the TNF-α group were significantly decreased (P <0.05) and the TM activity significantly decreased (P <0.05). However, there was no difference in TM protein level and activity between the BAY11-7082 control group and the control group (P <0.05). Conclusion TNF-α can significantly reduce TM expression and inhibit its activity, NF-κB pathway involved in this process.