【作者】 林芮禾；

【导师】 彭裕文； 李瑞锡； 俞彰； 王劼；

【作者基本信息】 复旦大学 ， 解剖与组织胚胎学， 2006， 博士

【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PGE₂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PGE₂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PGE₂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PGE₂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PGE₂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PGE₂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PGE₂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。更多还原

【Abstract】 γ-aminobutyric acid （GABA） is an important inhibitory neurotransmitter in nervous system and carries out inhibitory function by binding to GABA_A, GABA_B, GABA_C receptors. GABA_B receptors is a GTP-binding protein-coupled receptor （GPCR） consisting of GABA_BR1 and GABA_BR₂ subunits for the signal transduction.Physiological data showed that fine afferents （C and A_δ fibers） can be depolarized to elicit pain and release CGRP, SP or Glu. But the process can be inhibited by activating the presynaptic GABA_B receptors on the primary afferent fibers by the GABAergic interneurons in spinal dorsal horn regulating pain transmission. However, neither the characteristics of the afferent fibers that express GABA_B receptor nor the relationship between peptidergic and nonpeptidergic afferent fibers with GABAergic neurons in spinal dorsal horn are elucidated.Astrocytes have been demonstrated to be involved in pain regulation at the spinal level, they can also express GABA_B receptors. To further understand the role of GABA_B receptors in inflammatory pain transmission, we have investigated the phosphorylation and expression changes of GABA_B receptors in astrocytes.In the present study, we first investigated the neuronal subpopulations in the DRG which express the GABA_BRI by doublefluorescence immunohistochemistry. 94% of the peptidergic and 88% of the nonpeptidergic small size DRG neurons expressed GABA_BR.In the next step, we found CGRP and IB4 immunoreactive primary afferent terminals had close contacts with the GABA positive labeled interneurons in the spinal dorsal horn by doublefluorescence immunohistochemistry. Furthermore, some central terminals of type I and type II synaptic glomeruli were labeled by GABA_BR1in the superficial spinal dorsal horn as observed by electron microscopy. We also found some astrocytes labeled by GABAbR1 in the spinal dorsal horn. From these results we suggest that presynaptic GABA_BR may be involved in the modulation of nociceptive transmission.In the third part, we studied the phosphorylation of transcription factor cAMP response element binding protein （CREB） and GABA_BR2 in cultured astrocytes treated by prostaglandin E₂ （PGE₂） at different time points by immunocytochemistry and Western blot. The levels of phosphorylation of CREB and GABA_BR2 could be enhanced significantly by PGE2 in a time-dependent manner.Furthermore, we investigated the expression changes of GABA_B receptors in astrocytes under inflammatory situation. The results showed the mRNA expression levels of GABAbR1 and GABA_BR2 were down-regulated in a time-dependent manner by PGE2. Western blot analysis showed a reduced amount of GABAbR1b protein level compared to control values by the treatment of PGE2 for 24 h.Together our observations suggest the presynaptic GABA_B receptors may be involved in the modulation of nociceptive transmission. The changes of phosphorylation and expression of GABA_B receptors reflect its critical role in CNS inflammation and pain regulation.更多还原