辣椒素敏感的初级传入C纤维兴奋脊髓后角HAP1神经元的超微结构与生物化学证据

发布时间：2018-06-13 23:27

本文选题：亨廷顿蛋白相关蛋白1 + 辣椒素　；参考：《华中科技大学》2006年硕士论文

【摘要】： 亨廷顿蛋白相关蛋白1(huntingtin-associated protein 1,HAP1)是一种功能未明的蛋白质,因其与亨廷顿病(Huntington’s disease, HD)基因产物亨廷顿蛋白(huntingtin)具有相互作用而被确定为第一个亨廷顿蛋白相关蛋白。HAP1至少具有2种同工异构型(isoforms),即HAP1A和HAP1B。HAP1广泛分布于神经系统内,其中HAP1A主要定位于神经元胞体和轴突终末内,而HAP1B主要定位于神经元胞体和树突内。本实验室以前的研究发现,HAP1在大鼠脊髓背角浅层有高水平表达,而在脊髓腹角的表达水平极低,外周疼痛刺激可使脊髓背角浅层内HAP1及其mRNA表达水平升高,由此提示脊髓背角内的HAP1可能参与感觉信息特别是伤害性信息(痛觉)的初级传入和/或调控。为了证明这一假设,本研究应用免疫电镜技术观察初级传入C纤维(简称C纤维)与脊髓背角内HAP1神经元的突触联系,并应用免疫印迹和RT-PCR技术检测C纤维是否参与外周疼痛刺激促进脊髓背角内HAP1及其mRNA表达的过程。对正常大鼠腰段脊髓进行HAP1B的ABC法免疫组织化学染色,光镜下观察显示,HAP1免疫反应产物主要分布在脊髓灰质背角浅层神经元胞体和神经毡内,灰质其他各层无或极少HAP1B表达,白质内未见明显HAP1B免疫反应产物。经成年大鼠寰枕骨下穿刺蛛网膜下腔插管注射辣椒素到腰段脊髓特异性损毁C纤维,7天后取腰段脊髓进行HAP1B的ABC法免疫电镜标记。电镜下观察发现,在蛛网膜下腔注射辣椒素大鼠脊髓背角浅层内,大量变性的C纤维轴突终末表现为囊泡结构模糊不清,轴浆基质内电子密度均匀增高;HAP1B免疫反应产物主要分布在神经元胞体和树突内,并可见变性轴突终末与1至多个HAP1B阳性树突形成非对称性轴树突触或突触球。免疫印迹和RT-PCR检测显示,在新生期(出生后2天)皮下注射辣椒素的成年大鼠和成年期蛛网膜下腔注射辣椒素7天的大鼠,单侧疼痛刺激(足底注射10%福尔马林)24h后,注射侧脊髓背角内HAP1B和其mRNA的表达水平与对照侧无明显差异,而在新生期皮下注射溶媒的成年大鼠,疼痛刺激侧脊髓背角内HAP1B和其mRNA的表达水平均较对照侧明显增高。辣椒素是一种对C纤维具有选择性毒性神经毒素,在新生期皮下注射辣椒素或在成年期蛛网膜下腔注射辣椒素均可使C纤维大量溃变或变性,是一种公认的破环C纤维的工具药。本实验中,在蛛网膜下腔注射辣椒素的大鼠脊髓背角浅层内C纤维变性终末与HAP1B阳性的树突间非对称性的轴树突触或突触球的存在证明,脊髓背角中的HAP1B神经元能被C纤维传入的伤害性信号激活;疼痛刺激能促进脊髓背角中HAP1B和其mRNA的表达,而在辣椒素毁损C纤维后,疼痛刺激促进脊髓背角中HAP1B和其mRNA表达的作用不再存在或明显减弱,这现象则表明,C纤维参与外周疼痛刺激促进脊髓背角内HAP1及其mRNA表达的过程。脊髓背角中的HAP1在外周伤害性刺激通过C纤维的兴奋作用而表达增加后可能参与对伤害性信息传入的调节过程。
[Abstract]:Huntington protein related protein 1 (huntingtin-associated protein 1, HAP1) is an unfunctional protein that has been identified as the first Huntington protein related protein.HAP1 with at least 2 types of identical isomer because of its interaction with the Huntington disease (Huntington 's disease, HD) gene product Huntington protein (huntingtin). RMS), that is, HAP1A and HAP1B.HAP1 are widely distributed in the nervous system, in which HAP1A is located mainly in the neuronal cell body and the end of the axon, while HAP1B is located mainly in the neurons and dendrites. Previous studies in this laboratory found that HAP1 was highly expressed in the shallow layer of the dorsal horn of the spinal cord of the rat, and the expression level in the ventral horn of the spinal cord was very low, and the peripheral part of the spinal cord was very low. Pain stimulation can increase the level of HAP1 and mRNA in the superficial dorsal horn of the spinal cord, suggesting that HAP1 in the dorsal horn of the spinal cord may be involved in primary afferent and / or regulation of sensory information, especially nociceptive information (pain sensation). In order to prove this hypothesis, this study applied immuno electron microscopy to observe the primary afferent C fiber (C fiber) and the spinal cord. The synapse of HAP1 neurons in the dorsal horn and the immunoblotting and RT-PCR techniques were used to detect whether C fibers were involved in peripheral pain stimulation to promote the expression of HAP1 and mRNA in the dorsal horn of the spinal cord.
ABC immunohistochemical staining of HAP1B in the spinal cord of normal rats was carried out. The results of light microscopy showed that the products of HAP1 immunoreaction were mainly distributed in the shallow layer neurons of the spinal gray matter and the nerve felt in the shallow layer of the gray matter of the spinal cord. There was no or little HAP1B expression in the other layers of the gray matter, and there was no obvious HAP1B immunoreactivity in the white matter. Under the subarachnoid subarachnoid catheterization, capsaicin was injected into the lumbar spinal cord with specific damage to the C fiber, and the lumbar spinal cord was taken for HAP1B by ABC immunoelectron microscopy 7 days later. Under the electron microscope, it was found that in the subarachnoid injection of capsaicin in the superficial layer of the spinal dorsal horn of the capsaicin, a large number of denatured C fiber axons were characterized by the ambiguous structure of the vesicles. The electron density in the pulp matrix increased evenly, and the HAP1B immunoreaction products were mainly distributed in the neurons and dendrites, and the denatured axon terminals formed asymmetric axonal synapses or synapses with 1 to a number of HAP1B positive dendrites.
Western blot and RT-PCR showed that the expression level of HAP1B and mRNA in the dorsal horn of the spinal cord was not significantly different from the control side, but there was no significant difference in the expression level of the HAP1B and its mRNA in the dorsal horn of the spinal cord after the subcutaneous injection of capsaicin and the subarachnoid injection of capsaicin for 7 days in the adult rat and the subarachnoid injection of the subarachnoid cavity. In adult rats, the expression levels of HAP1B and mRNA in the spinal cord dorsal horn were significantly higher than those in the control side.
Capsaicin is a selective toxic neurotoxin for C fibers. Capsaicin injection in the newborn period, or capsaicin injection in the subarachnoid space of adulthood, can make C fiber a large amount of degeneration or denaturation. It is a recognized tool for breaking the C fiber. In this experiment, C in the shallow layer of the spinal dorsal horn of the subarachnoid injection of capsaicin in rats The presence of asymmetrical axisymmetric synapses or synapses between fiber degeneration and HAP1B positive dendrites proves that HAP1B neurons in the dorsal horn of the spinal cord can be activated by the nociceptive signals transmitted by C fibers; pain stimulation can promote the expression of HAP1B and mRNA in the dorsal horn of the spinal cord, and the pain stimulates the dorsal horn of the spinal cord after the paprika destroys the C fiber. The expression of HAP1B and its mRNA no longer exists or significantly diminished, which indicates that the involvement of C fibers in peripheral pain stimulates the expression of HAP1 and its mRNA in the dorsal horn of the spinal cord. HAP1 in the dorsal horn of the spinal cord may be involved in the regulation of nociceptive information after the increase of the expression of the peripheral nociceptive stimulation through the excitation of the C fiber. Process.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

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