人睫状肌细胞核因子κB在曲伏前列腺素作用下的变化

背景：核因子κB可能与葡萄膜巩膜房水流出通道的多种细胞信号调控有关。目的：观察前列腺素类曲伏前列腺素药物作用下，体外培养的人睫状肌细胞核因子KB及其抑制因子（inhibitor，IκB）的变化。设计、时间及地点：对比观察实验，于2005-03，2006-11在中山眼科中心实验室完成。材料：供体取自中山眼科医院，摘自死亡1h内无眼疾青年尸体眼球。患者家属对实验知情同意，并自愿捐献。方法：在人睫状肌细胞培养基中加1umol／L曲伏前列腺素，根据孵育时间的不同分为4组，即0h对照组和6，12，24h组。主要观察指标：采用real-time RT-PCR、免疫荧光半定量分析和ELISA法分别检测上述时间组核因子κBp65、IκBa在基因和蛋白水平的表达。结果：①与对照组比较，6，12，24h组核因子κBp65 mRNA表达均下降（F=17．068，P=0．001）：IκBαmRNA6h组、12h组较对照组改变不明显（P〉0．05），24h组较对照组表达增加（F=32．742，P=0．000）。②免疫荧光半定量分析表明：核因子KBp65荧光强度6，12，24h组均较对照组减少（F=17．216，P=0．000）；IκBQ6h组较对照组没有明显改变（P=0．134）、12h组较对照组轻微下降（P=0．032），24h组较对照组明显增加（F=17．346，P=0．001）。③ELISA法检测磷酸化核因子κBp65随药物作用时间延长而逐渐下降（F=15．4，P=0．001）。结论：曲伏前列腺素作用于人睫状肌细胞后，核因子κBp65的基因表达下调，核易位抑制，IκBα的基因表达上调。

Effect of travoprost on nuclear factor kappa B expression in human ciliary muscle cells

BACKGROUND: Nuclear factor kappa B (NF-κB) is possibly related to regulation of various cell signals that are derived from aqueous uveoscleral outflow pathway.OBJECTIVE: To explore effect of travoprost on the expression of NF-κB and inhibitor-κB (I-κB) in human ciliary muscle cells cultured in vitro. DESIGN, TIME AND SETTING: A contrast study, which was performed in the Laboratory of Zhongshan Ophthalmology Center from March 2005 to November 2006.MATERIALS: Eyeballs were obtained from the youth who died due to other diseases except eye disease no more than one hour. The relatives voluntarily provided the informed consent.METHODS: Travoprost (1 μmol/L) was added in human ciliary muscle cell culture medium, and then the samples were divided into four groups according to culture time, including 0-hour (control group), 6-hour, 12-hour, and 24-hour experimental groups. MAIN OUTCOME MEASURES: Expression of mRNA and protein of NF-κB p65 and I-κBα in the four groups by using real-time RT-PCR, immunofluorescence relative quantitative analysis and enzyme linked immunosorbent assay (ELISA) techniques. RESULTS: As compared to control group, mRNA expression of NF-κB p65 in the 6-hour, 12-hour, and 24-hour experimental groups was decreased (F=17.068, P=0.001); while mRNA expression of I-κBα was not changed remarkably in the 6-hour and 12-hour experimental groups (P > 0.05), but the expression was significantly higher than that in the 24-hour experimental group (F=32.742, P=0.000). Immunofluorescence relative quantitative analysis showed that the fluorescence intensity of NF-κB p65 in the 6-hour, 12-hour, and 24-hour experimental groups were weaker than that in the 0-hour control group (F=17.216, P=0.000); additionally, as compared to 0-hour control group, fluorescence intensity of I-κBα in the 6-hour experimental group was not changed remarkably (P=0.134), that in the 12-hour experimental group was weakened (P=0.032), and that in the 24-hour experimental group was strengthened (F=17.346, P=0.001). ELISA revealed that expression of phosphorylated NF-κB p65 was decreased gradually by the time of being induced by travoprost (F=15.4, P=0.001). CONCLUSION: Travoprost can down-regulate mRNA expression of NF-κB p65, inhibit nuclear translocation, and up-regulate mRNA expression of I-κBα in human ciliary muscle cells.