摘要
目的:了解视黄酸核受体α(RARα)对大鼠神经元功能的必要性。方法:采用组织消化原代贴壁法分离培养大鼠原代海马神经元,利用腺病毒载体特异沉默RARα;利用Real-Time PCR分析沉默RARα对神经元视黄酸(RA)信号各受体以及神经细胞标志物的影响;利用活细胞钙影像分析沉默RARα对神经元钙兴奋性的影响。结果:免疫荧光显示,分离培养的细胞90%表达神经元标志神经元特异性烯醇化酶(NSE),腺病毒转染效率可达80%。PCR结果显示,RARα沉默后RARα表达降低75%(P<0.01),其他受体均显著降低(P<0.01),但RARβ显著上调(P<0.05)。活细胞钙影像显示,沉默组钙兴奋性显著降低(P<0.05),全反式视黄酸(ATRA)预处理24h能显著增强钙兴奋性(P<0.01)。结论:RARα的缺失能显著降低原代海马神经元的神经元标志物NSE的表达,并显著损伤神经元的钙兴奋性。
Objective To study the necessary of retinoic acid receptor α(RARα) for rat neuron function.Methods Tissue digestion was used to isolate and cultivate the rat primary hippocampal neurons,and the adenovirus vector was used to specifically silence the RARα;Real-Time PCR was used to analyze the influence of silenced RARα in retinoic acid(RA) receptors and the markers of nerve cells;live cell imaging analysis was performed to analyze the influence of the calcium excitability of neurons silenced RARα.Results The immunofluorescence results showed that 90% of the isolated cells expressed the neuron marker neuron-specific enolase(NSE),the adenoviral transfection efficiency was up to 80%.The PCR results showed the expression of RARα in silenced RARα neuron was decreased by 75%(P<0.01),the other receptors were significantly decreased(P<0.01),but RARβ was significantly increased(P<0.05).The live cell calcium imaging results showed the calcium excitability in silent group was significantly reduced(P<0.05),however all-trans retinoic acid(ATRA) pretreatment for 24 h could significantly enhance the calcium excitability(P<0.01).Conclusion The absence of RARα can significantly reduce the neuron marker NSE expression of the primary hippocampal neurons,and significantly damage the neuronal calcium excitability.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2011年第6期976-980,964,共6页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金重点项目资助课题(30830106)
国家自然科学基金青年基金资助课题(81100454)
关键词
原代海马神经元
视黄酸
视黄酸核受体α
钙兴奋性
基因沉默
primary hippocampal neurons
retinoic acid
retinoic acid receptor α
calcium excitability
gene silence
