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目的研究脂多糖(LPS)诱导人角膜基质细胞核因子-κBp65(NF-κBp65)和NF-κB抑制因子-α(IκB-α)的表达及意义。方法原代培养人眼角膜基质细胞,用免疫细胞化学SABC方法进行细胞鉴定。采用绿脓杆菌LPS刺激角膜基质细胞,分别于刺激前(0 h),刺激后1、2、4、8 h收集细胞。Western blot检测胞浆内IκB-α蛋白及胞核内NF-κBp65蛋白的表达。结果与0 h相比,LPS刺激细胞1 h时,即可检测到胞核内NF-κBp65蛋白表达增加,其电泳条带的光密度值随着刺激时间的延长而逐渐变亮,4 h表达最高。LPS刺激细胞后各时点的NF-κBp65蛋白表达与0 h组比较,差异有统计学意义(P均<0.01)。LPS刺激角膜基质细胞后,在各时间点均可引起胞浆IκB-α表达下降,4 h时表达最低(P均<0.01)。结论LPS可引起人眼角膜基质细胞IκB-α的降解,促进NF-κB的核转位,在角膜炎的过程中可能起一定的抗炎作用。
Objective To investigate the expression of NF-κBp65 and IκB-α in human corneal stromal cells induced by lipopolysaccharide (LPS). Methods Human corneal stromal cells were primary cultured and identified by immunocytochemical SABC method. Corneal stromal cells were stimulated with Pseudomonas aeruginosa LPS, and cells were harvested before stimulation (0 h), 1, 2, 4 and 8 h after stimulation. Western blot was used to detect the expression of IκB-α protein and NF-κB p65 in the nucleus. Results Compared with 0 h, the expression of NF-κBp65 protein in nuclei was detected when LPS stimulated the cells for 1 h. The optical density of the electrophoresis band gradually lightened with the stimulation time prolonged, and reached its peak at 4 h highest. Compared with 0 h group, the expression of NF-κB p65 in LPS-stimulated cells at each time point was significantly different (all P <0.01). After LPS stimulated corneal stromal cells, the expression of IκB-α in cytoplasm was decreased at all time points, and the expression was lowest at 4 h (all P <0.01). Conclusion LPS can cause the degradation of IκB-α in human corneal stromal cells and promote the nuclear translocation of NF-κB, which may play an anti-inflammatory role in the process of keratitis.