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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 understand the necessity of retinoic acid nuclear receptor α (RARα) on neuronal function in rats. Methods: Primary cultured rat primary hippocampal neurons were isolated and cultured by tissue explant method and RARα was silenced by adenoviral vector. Real-time PCR was used to analyze the effect of silencing RARα on the expression of retinoic acid (RA) signal receptors and Neural cell markers; Calcium images of living cells were used to analyze the effect of silencing RARα on the neuronal calcium excitability. Results: Immunofluorescence showed that 90% of cultured cells expressed neuron-specific neuron-specific enolase (NSE), and the transfection efficiency of adenovirus was up to 80%. The results of PCR showed that the expression of RARα was decreased by 75% (P <0.01) after RARα silencing, while the other receptors were significantly decreased (P <0.01), but RARβ was significantly increased (P <0.05). Calcium in living cells showed that calcium excitability was significantly decreased in silencing group (P <0.05), and 24h pretreatment with all-trans retinoic acid (ATRA) significantly increased calcium excitability (P <0.01). CONCLUSION: The deletion of RARα can significantly reduce the expression of neuronal marker NSE in primary hippocampal neurons and significantly impair the calcium excitability of neurons.