吗啡处理后原代培养的海马神经元CREB_1蛋白的改变

Changes of CREB_1 protein in primary cultured hippocampal neurons treated with morphine

目的研究急、慢性吗啡处理后原代培养的海马神经元cAMP反应元件结合蛋白(CREB1蛋白)的改变。方法取原代培养7d的成熟海马神经元,随机分为吗啡急性作用组、纳洛酮急性戒断组、吗啡慢性作用组、纳洛酮慢性戒断组和空白对照组,每组6皿细胞。采用细胞免疫荧光染色法检测CREB1蛋白相对含量。结果吗啡急性作用组和纳洛酮急性戒断组原代培养的海马神经元内CREB1蛋白表达没有影响,而吗啡慢性作用组海马神经元内CREB1蛋白表达明显增强(P<0.01),且纳洛酮慢性戒断组免疫荧光强度进一步增加(P<0.01),但与吗啡慢性作用组比较差异无统计学意义。吗啡慢性作用组海马神经元内CREB1蛋白较吗啡急性作用组表达明显增强(P<0.01),纳洛酮慢性戒断组较急性戒断组CREB1蛋白表达差异有统计学意义(P<0.01)。结论慢性、多次使用成瘾药物才引起CREB1蛋白表达增加,其机制可能是多次使用吗啡通过反复对细胞内信号转导途径的影响引起神经元细胞核CREB1蛋白的改变。

Objective To investigate the changes of CREBa protein in primary cultured hippocampal neurons with morphine treatment. Methods The hippocampi were dissected from new-born Sprague-Dawley rats. Primary hippocampal neuronal cultures of 7 days in vitro were used and were divided randomly into 5 groups （n= 6 plate） ： acute morphine treatment group（A）, acute abstinent group（B）, chronic morphine treatment group（C）, chronic abstinent group（D） and control group（E）. Control group was treated with saline. The CREB1 protein levels were detected with immunofluorescence and laser scanning confocal microscope（LSCM） imaging techniques. Result In group A and B, CREB1 protein levels not changed compared with those in group E. In group C and D, CREB1 protein levels increased significantly compared with those in group E（P〈0. 01）, and CREB1 protein levels in group D not changed compared with those in group C. CREB1 protein levels in group C increased significantly compared with those in group A, and in group D increased significantly compared with those in group B（P〈0. 01）. Conclusion CREB1 protein levels increased significantly in primary cultured hippocampal neurons when chronic morphine treatment and abstinence, which might be a potential molecular mechanism of opioid tolerance and dependence, reflecting the changes of signal transduction when multiple uses of morphine.