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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 κB may be involved in the regulation of a variety of cellular signals in the aqueous humor outflow tract of uveal sclera. Objective: To observe the changes of nuclear factor кB and its inhibitor (ⅠκB) in human ciliary muscle cells cultured with prostaglandin travoprost. DESIGN, TIME AND SETTING: The comparative observation experiment was performed at the Zhongshan Ophthalmic Center Laboratory from March 2005 to November 2006. Material: The donor was taken from Zhongshan Ophthalmic Hospital, excerpted from the corpse eyeball without eye disease within 1h of death. Family members of patients informed consent and voluntary donation. Methods: Human ciliary muscle cell culture medium plus 1μmol / L travoprost, according to the different incubation time is divided into 4 groups, namely 0h control group and 6,12,24 h group. MAIN OUTCOME MEASURES: Real-time RT-PCR, semi-quantitative analysis of immunofluorescence and ELISA were used to detect the expression of nuclear factor κBp65 and IκBα at the gene and protein levels respectively. Results: ①Compared with the control group, the expression of nuclear factor κB p65 mRNA decreased at 6, 12, 24 hours (F = 17.068, P = 0.001); the expressions of IκBα mRNA in 6 hours and 12 hours group were not significantly changed (P> 0.05) Compared with the control group, the expression increased (F = 32.742, P = 0.000). ② Semiquantitative analysis of immunofluorescence showed that the fluorescence intensity of nuclear factor κBp65 was lower in the 6,12,24h group than in the control group (F = 17.216, P = 0.000); the IκBα6h group had no significant change compared with the control group (P = 0.134) Slightly lower than the control group (P = 0.032), 24h group increased significantly compared with the control group (F = 17.346, P = 0.001). (3) The level of phosphorylated nuclear factor kappa B p65 decreased gradually with the prolongation of drug effect by ELISA (F = 15.4, P = 0.001). CONCLUSION: Travoprost inhibits the gene expression of nuclear factor kappa B p65, inhibits nuclear translocation, and up-regulates the gene expression of I kappa B alpha in human ciliary muscle cells.