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Aquaporin4基因敲除对吗啡诱导条件性位置偏爱及抑制神经再生的影响

作者: 专业:药理学 导师:李锦 年度:2009 学位:硕士  院校: 南京医科大学

Keywords

opioid dependence, aquaporin 4, conditioned-place preference, neurogenesis

        水通道蛋白4(aquaporin-4, AQP4)是脑内含量最丰富、转运效率最高的水通道亚型,主要表达于星形胶质细胞和室管膜上皮细胞。AQP4不仅是脑内一种特异性的水转运孔道,而且可能调节星形胶质细胞的功能,广泛参与脑内微环境的维持、神经传导、神经再生等重要生理功能的调节。实验室前期研究发现,AQP4基因敲除能增强吗啡镇痛、减轻吗啡耐受和躯体依赖的形成。然而,AQP4是否参与了吗啡慢性处理所致的精神依赖过程尚未见文献报道。本文主要研究目的是探讨AQP4对吗啡精神依赖的调节作用及可能机制。在条件性位置偏爱(CPP)模型上,吗啡处理8天可使小鼠形成明显CPP。在吗啡10mg/kg剂量时,AQP4基因敲除能显著减轻吗啡慢性处理所致CPP效应的形成。在上述实验模型上,以反映细胞增殖的标志物5-溴脱氧尿嘧啶核苷(5-bromo-2’-deoxyuridine-5’-mono-phosphate,BrdU)掺入免疫组化方法观察吗啡依赖后神经再生的水平,结果发现野生型吗啡依赖小鼠海马齿状回BrdU阳性细胞数目显著下降约26%,但AQP4基因敲除动物BrdU阳性细胞数下降不显著,仅10%,提示AQP4基因敲除减轻吗啡慢性处理导致的海马神经干细胞增殖的抑制作用。应用免疫荧光双染法检测BrdU阳性细胞向神经元(NeuN)的分化,结果发现新生细胞大部分分化为神经元,AQP4基因敲除抑制吗啡慢性处理所致海马神经干细胞存活数量的减少,但是对存活率和新生细胞向神经元的分化率没有显著影响。此外,,吗啡慢性处理不影响两种基因型小鼠海马中细胞外信号调节激酶(phosphorylated extracellular signal-regulated kinase,ERK)的磷酸化水平。但是,吗啡慢性处理能降低野生型小鼠cAMP反应元件结合蛋白(cAMP respone element protein,CREB)磷酸化水平,AQP4基因敲除能显著减弱这一作用。提示AQP4基因敲除可能通过调节CREB的活性而减轻吗啡慢性处理对海马神经干细胞增殖的抑制作用,且不依赖ERK通路,其具体机制还有待进一步的研究。本文工作首次发现AQP4基因敲除减弱吗啡所致条件性位置偏爱的形成,其机制可能与其对海马神经再生的调节有关。
    In the central nervous system, aquaporin-4 (AQP4) is the most abundant isoform of water transporting channels and is localized in ependymal cells lining the ventricles and astrocyte membranes . AQP4 has gained much attraction due to its involvement in the regulation of the considerable physiologic function, including maintenance of the microenvironment, neuro-transmission, neurogenesis, and so on. Our previous study has shown that AQP4 deficiency could potentiate morphine analgesia, attenuate morphine tolerance and physical dependence. However, whether AQP4 particpates in the psychological dependence induced by morphine remains unclear. In the present study, the effects of AQP4 on the psychological dependence on morphine and the possible mechanism are investigated.In mouse conditioned-place preference (CPP) model, morphine (10 mg/kg, sc, 8d) induced the acquisition of CPP, whlie AQP4 knockout attenuated the acquisition of morphine-indece CPP. In the CPP model, we found that BrdU-positive cells number in hippocampus of morphine-dependent wild-type mice was down-regulated by 26% by using immunohistochemistry assay. But in morphine-dependent AQP4 knockout mice, the Brdu-positive cells number was down-regulated only by 10%. It suggested that AQP4 knockout attenuated the inhibition to the proliferation of hippocampal neural stem cells. The differentiation of newly formed cells was analyzed by double labeling for mature neurons with BrdU and neuron specific unclear protein (NeuN). We found that AQP4 knockout suppressed the decrease in quatity of hippocampal surviving BrdU-positive cells induced by chronic morphine treatment, but the survival rate had no significant change. In addition, the population of surviving BrdU-positive cells essentially matured into neurons, but the phenotypic expression pattern remained unchanged between groups. The phosphorylation of ERK1/2 and CREB in the hippocampus was also studied. We found that chronic morphine treatment did not markedly affect the phosphorylation of ERK1/2 in both wild-type and AQP4 knockout mice. However, chronic morphine treatment reduced the phosphorylation of CREB in wild-type mice, and AQP4 knockout attenuated the morphine-induced decrease in CREB phosphorylation. These results suggested that AQP4 knockout attenuating the inhibition to hippocampal neurogenesis induced by chronic exposure to morphine might be related to its modulating CREB phosphorylation.In conclusion, our studies demonstrated that AQP4 knockout attenuated the acquisition of morphine-induced CPP, and the mechanism might be related to its modulating hippocampal neurogenesis.
        

Aquaporin4基因敲除对吗啡诱导条件性位置偏爱及抑制神经再生的影响

英文缩略词表6-7
中文摘要7-9
英文摘要9-10
前言11-18
材料及方法18-26
结果26-35
讨论35-39
结论39-40
参考文献40-45
附录45-54
    在读期间发表或完成的论文45-46
    综述46-54
        参考文献50-54
    致谢54
        下载全文需50


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