GABA_B受体在脊髓水平痛觉调节中的作用

发布时间：2018-11-16 08:16

【摘要】： γ-氨基丁酸(GABA)是中枢神经系统中一种重要的抑制性神经递质，GABA_B受体是GABA受体家族中一类G蛋白偶联的代谢型受体。有生理功能的GABA_B受体由GABA_BR1和GABA_BR2构成。现已证明GABA_B受体参与了脊髓水平的痛觉调制，背根神经节神经元表达GABA_B受体。但目前对于背根神经节肽能和非肽能2个亚群小型神经元中GABA_B受体分布有何特点以及它们的中枢突末梢中的GABA_B受体是否受脊髓背角GABA能中间神经元的突触前抑制尚缺乏研究。同时，已知星形胶质细胞在脊髓水平痛觉调制中起了重要作用，可以感知中枢突末梢释放的各类炎症因子而发生功能变化，脊髓星形胶质细胞也呈GABA_B受体免疫阳性。然而，星形胶质细胞中GABA_B受体在炎症环境下是否发生磷酸化、转录和翻译等方面的变化，从而参与脊髓水平痛觉调制也未见报道。针对以上问题，本课题做了以下四方面的工作：一、以荧光双标记免疫组织化学方法，研究了两个不同亚群神经元中枢突在脊髓背角的分布特点。分别通过抗GABA_BR1和CGRP抗体以及抗GABA_BR1和IB4抗体分别进行双标，在激光共聚焦显微镜下观察GABA_BR1在2个不同亚群背根神经节神经元胞体以及中枢突的分布特点和表达差异。结果显示94％的肽能和88％的非肽能亚群的小型神经元均表达GABA_BR1，这些受体存在于神经元的胞体，其分布在脊髓背角特定板层的中枢突中也可能存在GABA_B受体。该结果表明在痛觉调制过程中，，背根神经节肽能和非肽能2个亚群的小型神经元都可能通过GABA_B受体在脊髓水平痛觉调制中发挥作用，但作用于脊髓背角的不同板层。二、用荧光双标记免疫组织化学和免疫电镜方法，在激光共聚焦显微镜下观察了正常大鼠CGRP和IB4阳性背根节神经元中枢突和脊髓背角GABA能中间神经元之间的联系，并研究脊髓背角浅层GABA_B受体免疫阳性的背根神经节神经元中枢突的超微结构。结果显示大部分脊髓背角GABA能神经元都表达GABA_B受体，CGRP免疫阳性和IB4阳性背根节神经元中枢突和脊髓背角GABA能中间神经元之间有密切关系；电镜下许多胶状质中突触小球的中央末梢为GABA_B受体免疫阳性，并作为突触前或突触后成分与周围末梢之间形成对称性和非对称性突触。同时也发现部分星形胶质细胞呈GABA_B受体免疫阳性。提示脊髓背角GABA能中间神经元可能通过分布在背根节神经元初级传入末梢上的GABA_B受体产生突触前抑制，参与脊髓水平的痛觉调制。三、采用体外培养、免疫细胞化学和Western blot等方法，研究了PGE_2刺激对体外培养的大鼠星形胶质细胞cAMP反应元件结合蛋白(cAMP responseelement binding protein，CREB)和GABA_BR2(Ser892位点)磷酸化水平的影响。结果表明经10μM的PGE_2刺激后，大鼠星形胶质细胞中CREB磷酸化水平的变化与时间有一定的相关性，与对照组之间差异显著。提示PGE_2能通过提高大鼠星形胶质细胞转录因子CREB的磷酸化水平，调控基因转录，进而引发一系列下游反应。同时，经10μM的PGE_2刺激不同时间后，星形胶质细胞GABA_BR2(Ser892)磷酸化水平明显增高，并具有时间依赖性。结果提示炎症环境下大鼠星形胶质细胞CREB和GABA_BR2(Ser892)磷酸化水平增高，可能参与细胞生理活动的调节。四、采用体外培养、RT-PCR和Western blot等方法研究了不同时间点一定剂量的PGE_2对体外培养的大鼠星形胶质细胞GABA_BR1和GABA_BR2转录和翻译水平的影响。结果表明经10μM的PGE_2刺激后，大鼠星形胶质细胞中转录和翻译水平与对照组之间相比明显下调，差异显著，并有时间依赖性。提示一定剂量的PGE_2刺激能引起大鼠星形胶质细胞GABA_B受体mRNA和蛋白水平发生变化，可能影响了炎症环境下大鼠星形胶质细胞的生理状态。综上所述，研究结果表明背根神经节中枢突末梢与脊髓背角GABA能中间神经元之间具有突触前抑制的形态学基础，能通过突触前GABA_B受体参与脊髓水平的痛觉调制；同时，在炎症环境下，星形胶质细胞中的GABA_B受体磷酸化，转录及翻译发生了一系列时间依赖性的变化。提示炎症痛中GABA_B受体可能通过多种途径参与脊髓水平痛觉调制。
[Abstract]:GABA-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system, and the GABA _ B receptor is a type of G-protein-coupled metabotropic receptor in the GABA receptor family. The physiological function of the GABA _ B receptor is composed of GABA _ BR1 and GABA _ BR2. It has been shown that the GABA _ B receptor is involved in the pain modulation of the level of the spinal cord, and the GABA _ B receptor is expressed in the dorsal root ganglion neurons. However, there is a lack of research on the characteristics of the distribution of the GABA _ B receptors in the 2 subpopulations of the dorsal root ganglion and the non-peptide, and whether the GABA _ B receptor in the central process of the dorsal root ganglion is affected by the presynaptic inhibition of the interneurons in the dorsal horn of the spinal cord. At the same time, it is known that astrocytes play an important role in the horizontal pain modulation of the spinal cord, and can sense the function changes of various inflammatory factors released by the central process tip, and the spinal-like astrocytes are also immunopositive for GABA _ B receptors. However, the presence of the GABA _ B receptor in astrocyte in the inflammatory environment has not been reported in the presence of phosphorylation, transcription and translation of the GABA _ B receptor. In view of the above problems, the subject has done the following four tasks: 1. The central process of two different subcluster neurons was studied by means of fluorescent double-labeled immunohistochemistry. The distribution of the dorsal horn of the spinal cord was characterized in that the GABA _ BR1 and the CGRP antibody and the anti-GABA _ BR1 and the IB4 antibodies were respectively double-labeled, and the neuronal cell and the central process of the GABA _ BR1 in the dorsal root ganglion of two different subpopulations were observed under the laser confocal microscope. The results showed that 94% of the peptides and 88% of the small neurons of the non-peptide subpopulations express GABA _ BR1, which are present in the cell of the neuron, which may be distributed in the central process of the specific lamina of the dorsal horn of the spinal cord. The results show that in the process of hyperalgesia, both the dorsal root ganglion peptide and the non-peptide can play a role in the horizontal pain modulation of the spinal cord through the GABA _ B receptor, but the effect The CGRP and IB4-positive dorsal root ganglion neurons in the normal rats were observed under the laser confocal microscope by means of fluorescent double-labeled immunocytochemical and immunoelectron microscopy. The relationship between the GABA and the middle neuron in the dorsal horn of the spinal cord and the study of the superficial GABA _ B receptor in the dorsal horn of the spinal cord The ultrastructure of the central process of the immune-positive dorsal root ganglion neurons showed that the GABA-B receptor, the CGRP-positive and the IB4-positive dorsal root ganglion neurons of the spinal dorsal horn of the spinal cord were closely related to the central neurons of the central and spinal dorsal horn of the dorsal root ganglion of the spinal cord. The central tip of the contact ball is an immunopositive of the GABA _ B receptor and acts as a presynaptic or postsynaptic The formation of symmetries and asymmetric synapses between the component and the peripheral tip. The GABA _ B receptors in the dorsal root of the dorsal root ganglion may be distributed through the GABA _ B distributed on the primary afferent terminal of the dorsal root ganglion neurons. 3. In vitro culture, immunocytochemistry and Western blot, the cAMP response element binding protein (cAMP response element binding protein) of the rat astrocyte cultured in vitro was studied by the method of in vitro culture, immunocytochemistry and Western blot. The effect of CREB and the phosphorylation of GABA _ BR2 (Ser892 site). The results showed that after the 10. m The change of the level of the phosphorylation of REB was related to the time, and the difference between the control group and the control group was significant. The phosphorylation level of CREB, the transcription of regulatory genes, and the initiation of a series of downstream reactions. At the same time, after 10. m u.M of PGE _ 2, the astrocyte GA The level of phosphorylation of BA _ BR2 (Ser892) was significantly higher and time-dependent. The phosphorylation of BR2 (Ser892) was increased, and it was possible to take part in the regulation of cellular physiological activities. The effect of _ 2 on the transcription and translation of GABA _ BR1 and GABA _ BR2 in rat astrocytes cultured in vitro. The results showed that the PGE _ 2 was stimulated by 10. m The level of transcription and translation in the rat astrocyte was significantly reduced compared with that in the control group, and the difference was significant and time-dependent. It was suggested that a certain dose of PGE _ 2 stimulation could cause the rat astrocyte GA. The changes of the mRNA and protein levels of the BA _ B receptor may affect the physiological state of the rat astrocytes in the inflammatory environment. In conclusion, the results show that the central process tip of the dorsal root ganglion and the dorsal horn of the spinal cord can be in the middle The morphological basis of presynaptic inhibition between the neurons can be involved in the level of the spinal cord through the presynaptic GABA _ B receptor. Hyperalgesia; at the same time, in the inflammatory environment, the GABA _ B receptor phosphate in the astrocytes
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329;R33

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