GABA_B受體在脊髓水平痛覺調(diào)節(jié)中的作用
發(fā)布時(shí)間：2018-11-16 08:17
【摘要】： γ-氨基丁酸(GABA)是中樞神經(jīng)系統(tǒng)中一種重要的抑制性神經(jīng)遞質(zhì)，GABA_B受體是GABA受體家族中一類G蛋白偶聯(lián)的代謝型受體。有生理功能的GABA_B受體由GABA_BR1和GABA_BR2構(gòu)成�，F(xiàn)已證明GABA_B受體參與了脊髓水平的痛覺調(diào)制，背根神經(jīng)節(jié)神經(jīng)元表達(dá)GABA_B受體。但目前對于背根神經(jīng)節(jié)肽能和非肽能2個(gè)亞群小型神經(jīng)元中GABA_B受體分布有何特點(diǎn)以及它們的中樞突末梢中的GABA_B受體是否受脊髓背角GABA能中間神經(jīng)元的突觸前抑制尚缺乏研究。同時(shí)，已知星形膠質(zhì)細(xì)胞在脊髓水平痛覺調(diào)制中起了重要作用，可以感知中樞突末梢釋放的各類炎癥因子而發(fā)生功能變化，脊髓星形膠質(zhì)細(xì)胞也呈GABA_B受體免疫陽性。然而，星形膠質(zhì)細(xì)胞中GABA_B受體在炎癥環(huán)境下是否發(fā)生磷酸化、轉(zhuǎn)錄和翻譯等方面的變化，從而參與脊髓水平痛覺調(diào)制也未見報(bào)道。針對以上問題，本課題做了以下四方面的工作： 一、以熒光雙標(biāo)記免疫組織化學(xué)方法，研究了兩個(gè)不同亞群神經(jīng)元中樞突在脊髓背角的分布特點(diǎn)。分別通過抗GABA_BR1和CGRP抗體以及抗GABA_BR1和IB4抗體分別進(jìn)行雙標(biāo)，在激光共聚焦顯微鏡下觀察GABA_BR1在2個(gè)不同亞群背根神經(jīng)節(jié)神經(jīng)元胞體以及中樞突的分布特點(diǎn)和表達(dá)差異。結(jié)果顯示94％的肽能和88％的非肽能亞群的小型神經(jīng)元均表達(dá)GABA_BR1，這些受體存在于神經(jīng)元的胞體，其分布在脊髓背角特定板層的中樞突中也可能存在GABA_B受體。該結(jié)果表明在痛覺調(diào)制過程中，背根神經(jīng)節(jié)肽能和非肽能2個(gè)亞群的小型神經(jīng)元都可能通過GABA_B受體在脊髓水平痛覺調(diào)制中發(fā)揮作用，但作用于脊髓背角的不同板層。 二、用熒光雙標(biāo)記免疫組織化學(xué)和免疫電鏡方法，在激光共聚焦顯微鏡下觀察了正常大鼠CGRP和IB4陽性背根節(jié)神經(jīng)元中樞突和脊髓背角GABA能中間神經(jīng)元之間的聯(lián)系，并研究脊髓背角淺層GABA_B受體免疫陽性的背根神經(jīng)節(jié)神經(jīng)元中樞突的超微結(jié)構(gòu)。結(jié)果顯示大部分脊髓背角GABA能神經(jīng)元都表達(dá)GABA_B受體，CGRP免疫陽性和IB4陽性背根節(jié)神經(jīng)元中樞突和脊髓背角GABA能中間神經(jīng)元之間有密切關(guān)系；電鏡下許多膠狀質(zhì)中突觸小球的中央末梢為GABA_B受體免疫陽性，并作為突觸前或突觸后成分與周圍末梢之間形成對稱性和非對稱性突觸。同時(shí)也發(fā)現(xiàn)部分星形膠質(zhì)細(xì)胞呈GABA_B受體免疫陽性。提示脊髓背角GABA能中間神經(jīng)元可能通過分布在背根節(jié)神經(jīng)元初級傳入末梢上的GABA_B受體產(chǎn)生突觸前抑制，參與脊髓水平的痛覺調(diào)制。 三、采用體外培養(yǎng)、免疫細(xì)胞化學(xué)和Western blot等方法，研究了PGE_2刺激對體外培養(yǎng)的大鼠星形膠質(zhì)細(xì)胞cAMP反應(yīng)元件結(jié)合蛋白(cAMP responseelement binding protein，，CREB)和GABA_BR2(Ser892位點(diǎn))磷酸化水平的影響。結(jié)果表明經(jīng)10μM的PGE_2刺激后，大鼠星形膠質(zhì)細(xì)胞中CREB磷酸化水平的變化與時(shí)間有一定的相關(guān)性，與對照組之間差異顯著。提示PGE_2能通過提高大鼠星形膠質(zhì)細(xì)胞轉(zhuǎn)錄因子CREB的磷酸化水平，調(diào)控基因轉(zhuǎn)錄，進(jìn)而引發(fā)一系列下游反應(yīng)。同時(shí)，經(jīng)10μM的PGE_2刺激不同時(shí)間后，星形膠質(zhì)細(xì)胞GABA_BR2(Ser892)磷酸化水平明顯增高，并具有時(shí)間依賴性。結(jié)果提示炎癥環(huán)境下大鼠星形膠質(zhì)細(xì)胞CREB和GABA_BR2(Ser892)磷酸化水平增高，可能參與細(xì)胞生理活動的調(diào)節(jié)。 四、采用體外培養(yǎng)、RT-PCR和Western blot等方法研究了不同時(shí)間點(diǎn)一定劑量的PGE_2對體外培養(yǎng)的大鼠星形膠質(zhì)細(xì)胞GABA_BR1和GABA_BR2轉(zhuǎn)錄和翻譯水平的影響。結(jié)果表明經(jīng)10μM的PGE_2刺激后，大鼠星形膠質(zhì)細(xì)胞中轉(zhuǎn)錄和翻譯水平與對照組之間相比明顯下調(diào)，差異顯著，并有時(shí)間依賴性。提示一定劑量的PGE_2刺激能引起大鼠星形膠質(zhì)細(xì)胞GABA_B受體mRNA和蛋白水平發(fā)生變化，可能影響了炎癥環(huán)境下大鼠星形膠質(zhì)細(xì)胞的生理狀態(tài)。 綜上所述，研究結(jié)果表明背根神經(jīng)節(jié)中樞突末梢與脊髓背角GABA能中間神經(jīng)元之間具有突觸前抑制的形態(tài)學(xué)基礎(chǔ)，能通過突觸前GABA_B受體參與脊髓水平的痛覺調(diào)制；同時(shí)，在炎癥環(huán)境下，星形膠質(zhì)細(xì)胞中的GABA_B受體磷酸化，轉(zhuǎn)錄及翻譯發(fā)生了一系列時(shí)間依賴性的變化。提示炎癥痛中GABA_B受體可能通過多種途徑參與脊髓水平痛覺調(diào)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
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R329;R33
本文編號：2334990
[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
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R329;R33
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 馮宇鵬,楊鯤,李云慶;大鼠延髓背角淺層內(nèi)CGRP陽性終末與GABA及甘氨酸能神經(jīng)元的突觸聯(lián)系[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2002年08期
本文編號：2334990
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