人睫状肌细胞核因子κB在曲伏前列腺素作用下的变化(英文)

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

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кB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1μmol/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、ⅠκBα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κB p65 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κBp65荧光强度6,12,24h组均较对照组减少(F=17.216,P=0.000);IκBα6h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。

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 μ mol/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 l-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 NF4cB p65 in the 6-hour, 12-hour, and 24-hour experimental groups was decreased （F=17.068, P=0.001）; while mRNA expression of l-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-κB α in human ciliary muscle cells.