白介素-1β诱导人脐静脉内皮细胞表达纤溶酶原激活物抑制剂-1的实验研究

Interleukin-1 beta induced plasminogen activator inhibitor-1 expression in human umbilical vein endothelial cells

目的:探讨白介素-1β(IL-1β)在诱导人脐静脉内皮细胞表达纤溶酶原激活物抑制剂-1(PAI-1)中的作用及机制。方法:培养原代人脐静脉内皮细胞,四甲基偶氮唑盐微量酶反应比色法监测不同浓度IL-1β刺激下的细胞活性;Real TimePCR及蛋白质印迹检测不同时间及不同浓度的IL-1β对PAI-1基因及蛋白表达的影响;进一步用核转录因子-κΒ(NF-κB)拮抗剂PDTC(5mol/L)探讨信号传导机制,检测IL-1β对PAI-1的诱导情况。结果:MTT结果示IL-1β在0～10ng/mL浓度下,细胞的活性不受影响(P>0.05),20ng/mLIL-1β刺激下细胞活性改变(P<0.05),差异有统计学意义;不同浓度IL-1β刺激下人脐静脉内皮细胞内PAI-1基因及蛋白的表达成时间依赖性,6h后开始增高(P<0.05),12h后达峰值(P<0.01),24h后开始下降(P<0.05),差异有统计学意义;IL-1β对PAI-1的刺激作用亦呈剂量依赖性,随着浓度(0、0.1、1、10ng/mL)的增加,PAI-1基因及蛋白的表达亦明显增加(P<0.05),差异有统计学意义;进一步的信号通路研究实验结果显示NF-κΒ拮抗剂PDTC可抑制IL-1β的诱导作用(P<0.05)。结论:在人脐静脉内皮细胞中,IL-1β可通过NF-κΒ信号途径上调PAI-1的表达,PDTC可抑制其上调作用。

Objective To investigate the effects and mechanism of interleukin-1 beta （IL-1β） on the expression of plasminogen activator inhibitor-1 （PAI-1） in human umbilical vein endothelial cells （HUVECs）. Methods MTF assay were performed to evaluate the cell viability of IL-1β induced primary cultured HUVECs. Real-time PCR and Western blot analysis were used to detect the expression of PAI-1 in IL-1β stimulated HUVECs. Nuclear factor-kappa B （NF-κB） inhibitor PDTC to explore the signaling mechanisms of IL-1βon the expression of PAI-1 in HUVECs. Results The MTT results showed that the cytoactive of HUVECs did not changed by using 0-10 ng/mL of IL-1β （P 〉 0.05 ）, but it changed when treated with 20 ng/mL IL-1β （P 〈 0.05 ）. The mRNA and protein expression of PAI-1 in IL-1β stimulated HUVECs was in a time-and dose-dependent manner. PAI-1 expression increased in 6 h （P 〈 0.05） and reached peak in 12 h （P 〈 0.01） then decreased in 24 h （P 〈 0.05） after IL-1β treatment, NF-κB inhibitor PDTC could restrain the effects of IL-1β. Conclusion IL-1β can induce the expression of PAI-1 in HUVECs through the NF-κB signal pathway.