纳洛酮对离体人脑神经元神经特异性烯醇化酶的影响

The effects of naloxone on neuron specific enolase in neurons in vitro

目的探讨不同浓度纳洛酮对体外培养人脑神经元缺氧损伤后细胞内外神经特异性烯醇化酶(NSE)浓度及NSE信使核糖核酸(mRNA)表达量变化的影响。方法应用无血清方法原代培养人胚胎大脑神经元,经免疫组化染色证实神经元比例达80%以上,对神经元进行缺氧结合无糖处理。将24孔培养板上的细胞随机分为单纯缺氧1 h组、缺氧+纳洛酮0.25μg/ml组、缺氧+纳洛酮5μg/ml组、缺氧+纳洛酮10μg/ml组和对照组,每组6孔细胞。缺氧处理1 h后复氧,于复氧24 h应用酶联免疫吸附试验(ELISA)测定细胞内外NSE浓度。将25 ml培养瓶内的细胞随机分为单纯缺氧0.5 h组、缺氧+纳洛酮0.5μg/ml组和缺氧+纳洛酮10μg/ml组,缺氧处理0.5 h后复氧,应用逆转录-聚合酶链反应(RT-PCR)观察缺氧及复氧后不同时间细胞内NSE mRNA表达量的变化。结果①单纯缺氧1 h组细胞外液NSE浓度明显升高(P<0.01),给药组随纳洛酮浓度增加,NSE浓度呈现逐渐降低趋势,至5μg/ml恢复至对照组水平。②单纯缺氧1 h组细胞内NSE浓度明显降低(P<0.01),给药组随纳洛酮浓度增加,细胞内NSE浓度呈现逐渐升高趋势,至10μg/ml恢复至对照组水平。③单纯缺氧0.5 h组,缺氧后NSE mRNA表达量明显升高(P<0.01),其后逐渐降低,并于复氧24 h后恢复至接近缺氧前水平。给予纳洛酮0.5μg/ml,NSE mRNA表达量在缺氧0.5 h后即刻仍明显升高。给予纳洛酮10μg/ml,NSE mRNA表达高峰延迟至复氧6 h出现。结论纳洛酮可减轻细胞膜损伤,减少细胞内酶的外漏,并在转录水平调节细胞NSE的表达,发挥对神经元缺氧性损伤的保护作用。

Objective To evaluate the effects of naloxone on neuron specific enolase（NSE） in combined oxygen-glucose deprivation of embryonic human cortical neurons. Methods The mixed cell culture rich in neurons prepared from embryonic human brains were deprived of both oxygen and glucos. ELISA and RT-FCR methods were used to detect the changes of intracellular and extracellular concentrations of NSE and the expression of NSE mRNA in hypoxic neurons treated with different concentrations of naloxone. Results The extracellular levels of NSE increased significantly after 1 h of oxygen-glucose deprivation（P〈0. 01）. The concentrations of NSE presented a dose-dependant decrease with the increasing of naloxone. One hour of oxygen-glucose deprivation was associated with a significantly decrease of intracellular NSE （P〈0. 01）. When treated with naloxone, the intracellular level of NSE presented a dose dependant increase and went back to control level at naloxone 10 ug/ml. Mild hypoxia injury for 30 min, the expression of NSE mRNA of neurons increased significantly （P〈0. 01 ）. After treated with different concentrations of naloxone, the peak value of each group were delayed to appear and went down with the increase of naloxone concentration. All of these parameters had significantly differences with control group （P〈0. 01）. Conclusion The results indicate that naloxone plays a protective role in neuron ischemic injury, which may be related to the protection of cell membrane and the regulation of NSE expression at transcriptional level.