谷氨酸、NMDA和吗啡对培养大鼠星星胶质细胞内钙振荡的影响

发布时间：2018-10-13 11:12

【摘要】：在中枢神经系统(CNS)中,胶质细胞的分布最为广泛。传统的观点认为胶质细胞只起着支持和填充作用,随着对胶质细胞研究的逐步深入,近年来形成了新的研究观点,即胶质细胞可以参与脑内突触的形成、神经递质的分泌和摄取,从而对神经元信息传递进行调控。胶质细胞对神经元信息传递的调控对正常和病理性脑功能(如药物成瘾等)具有重要的作用。星形胶质细胞(astrocyte)作为胶质细胞的一种亚型,在CNS中的作用也得到了越来越多的关注。不同于神经元兴奋后可产生动作电位,胶质细胞的兴奋性主要表现在胞内Ca2+浓度的升高,兴奋后的星形胶质细胞还可以释放胶质递质,胶质递质可以反作用于神经元,从而调控突触的功能及其可塑性,所以关于星形胶质细胞内Ca2+变化的研究成为了近年来的研究热点。本实验通过观察Sprague-Dawley (SD)大鼠大脑皮层原代培养星形胶质细胞在谷氨酸、NMDA、吗啡作用下细胞内Ca2+的变化的情况,目的是要了解神经元突触和星形胶质细胞之间发生相互作用的受体途径和阿片类药物对星形胶质细胞的直接影响。实验选取出生1-3天的SD大鼠,无菌条件下分离、纯化大鼠大脑皮层的星形胶质细胞,培养出纯度达95%以上的星形胶质细胞(GFAP染色法鉴定)。以Fura-2/AM为Ca2+指示剂,观察谷氨酸、NMDA、吗啡分别灌流前后培养的星形胶质细胞内Ca2+的变化。实验还进一步观察分别用代谢型谷氨酸受体5(mGluR5)、含NR2A和NR2B亚基的NMDA受体(NMDA receptor, NMDAR)、阿片μ受体的拮抗剂MTEP、NVP-AAM077和Ro25-6981、CTAP孵育星形胶质细胞,以阻断mGluR5、 NMDAR和阿片μ受体后对诱导的胞内钙振荡的影响。实验现象显示,谷氨酸、NMDA、吗啡分别灌流Fura-2/AM加载后的星形胶质细胞,荧光值F340/F380明显升高,这就说明,这三种药物均可明显升高细胞[Ca2+]i,而将其相应受体拮抗后,星形胶质细胞[Ca2+]i的升高幅度分别较只灌流谷氨酸、NMDA、吗啡明显降低,这就说明将相应受体拮抗后,[Ca2+]i升高的现象可以被显著抑制。实验结果： 1.谷氨酸可诱发星形胶质细胞内游离钙的浓度([Ca2+]i)升高； 2.谷氨酸通过激活mGluR5和NMDAR诱发星形胶质细胞[Ca2+]i升高； 3.吗啡通过激活阿片μ受体诱发星形胶质细胞[Ca2+]i升高。结论：实验结果表明,在离体培养的大鼠星形胶质细胞膜上存在mGluR5、含NR2A和NR2B亚基的NMDAR,以及阿片μ受体,这些受体的激活可以升高星形胶质细胞的[Ca2+]i,且这些受体依赖的[Ca2+]i的调控机制可能是星形胶质细胞与神经元交互作用的重要途径之一。
[Abstract]:In the central nervous system (CNS), glial cells are most widely distributed. The traditional view is that glial cells only play a supporting and filling role. With the gradual development of glial cell research, a new research view has been formed in recent years, that is, glial cells can participate in the formation of synapses in the brain. Neurotransmitters are secreted and ingested to regulate the transmission of neuronal information. Glial regulation of neuronal information transmission plays an important role in normal and pathological brain function, such as drug addiction. As a subtype of glial cells, astrocyte (astrocyte) has attracted more and more attention in CNS. The excitability of glial cells is mainly characterized by the increase of intracellular Ca2 concentration, and the release of glial transmitters by the excited astrocytes, which can counteract the neurons. In order to regulate synaptic function and plasticity, the study on the changes of Ca2 in astrocytes has become a hot topic in recent years. In this study, we observed the changes of Ca2 in primary cultured astrocytes of Sprague-Dawley (SD) rats induced by glutamate and NMDA, morphine. The aim of this study was to understand the receptor pathway that interacts with astrocytes and the direct effects of opioid drugs on astrocytes. The astrocytes of the cerebral cortex of SD rats were isolated and purified under aseptic condition. The astrocytes with purity of more than 95% were cultured (identified by GFAP staining). The changes of Ca2 in cultured astrocytes before and after administration of glutamate and NMDA, morphine were observed with Fura-2/AM as Ca2 indicator. The astrocytes were incubated with metabotropic glutamate receptor 5 (mGluR5), NMDA receptor (NMDA receptor, NMDAR), opioid 渭 receptor MTEP,NVP-AAM077 and Ro25-6981,CTAP containing NR2A and NR2B subunits, respectively. The effect of blocking mGluR5, NMDAR and opioid 渭 receptor on intracellular calcium oscillation was studied. The experimental results showed that the fluorescence value of F340/F380 was significantly increased in astrocytes after Fura-2/AM loading with glutamic acid and NMDA, morphine, which indicated that these three drugs could significantly increase [Ca2] I, but antagonize the corresponding receptors. The increase of [Ca2] I in astrocytes was significantly lower than that of glutamate and NMDA, morphine, respectively, which indicated that the increase of [Ca2] I could be significantly inhibited after antagonizing the corresponding receptor. Experimental results: 1. Glutamate could induce the increase of free calcium concentration ([Ca2] I) in astrocytes. Glutamate induced the increase of [Ca2] I in astrocytes by activating mGluR5 and NMDAR. Morphine induces the elevation of [Ca2] I in astrocytes by activating opioid 渭 receptors. Conclusion: the results indicate that there is NMDAR, and opioid 渭 receptor in mGluR5, containing NR2A and NR2B subunits on the cultured astrocytes in vitro. The activation of these receptors can increase [Ca2] I of astrocytes, and the regulatory mechanism of [Ca2] I dependent on these receptors may be one of the important pathways of astrocyte-neuron interaction.
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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