核转录因子κB在TNF-α诱导人气道上皮细胞hBD-2表达中的作用

Roles of NF-κB in expression of hBD-2 mRNA in human primary epithelial cells induced by TNF-α

目的观察肿瘤坏死因子-α(TNFα-)诱导人气道上皮细胞人β防御素-2(hBD-2)的表达及核转录因子κB(NFκ-B)在hBD-2表达中的作用。方法用TNFα-和或NFκ-B抑制剂PDTC处理体外培养人气道原代上皮细胞,RT-PCR法检测hBD-2 mRNA的表达,W estern b lot检测细胞质IκBα-蛋白活性变化,凝胶迁移试验(EMSA)检测不同时相NFκ-B活性的变化。结果TNFα-刺激0.5 h后可见人气道上皮细胞hBD-2 mRNA表达,并呈时间依赖性;PDTC可抑制hBD-2mRNA表达;NFκ-B在TNFα-刺激1 h后明显活化,抗体超迁移率实验结果显示p65-p50异型二聚体NFκ-B参与了NFκ-B的活化。结论一定剂量的TNFα-可诱导人气道上皮细胞hBD-2 mRNA表达,NFκ-B在TNFα-诱导气道上皮细胞hBD-2mRNA起重要的调控作用。

Objective To explore the roles of NF-κB in expression of human 13-defensin-2 （hBD-2） mR- NA induced by TNF-α in the human airway primary epithelial cells. Methods After the human bronchial primary epithelial cells were stimulated with TNF-α or first with NF-κB inhibitor PDTC, then TNF-α, the expression of hBD-2 mRNA was detected by RT-PCR. The IκB-α protein level in the cytoplasm was detected by Western blotting and the nuclear factor-kappa B （NF-κB） binding activity was analyzed by electrophoretie mobility shift assays. Results The hBD-2 mRNA could be detected after 2.5 h of TNF-α stimulation and expressed in a dose-dependent manner. The NF-κB could be activated after 0.5 h of TNF-α stimulation. The supershifts assays indicated that the p65 - p50 heterodimer formed complexes of NF-κB were involved in the activated of NF- κB. Oonclusion TNF-α can induce the expression of hBD-2 mRNA in a dose-dependent manner. The p65 - p50 heterodimer formed complexes of NF-κB play an important role in the regulation of hBD-2 gene expression in response to TNF-α.