CGRP对螺旋神经节细胞谷氨酸兴奋性毒性的干预作用

发布时间：2019-01-24 21:27

【摘要】： 螺旋神经节细胞是听觉传导通路的第一级神经元,其接受毛细胞释放的兴奋性递质谷氨酸的刺激产生兴奋性电位,传导至延髓脑桥连接处的蜗神经腹核和背核。但在某些病理情况下,如：噪音、缺血、缺氧、感染、耳毒性药物使用等,均可以引起谷氨酸的蓄积,通过多种途径造成螺旋神经节细胞的损伤,导致不可逆的听力损伤。传出神经系统通过神经通路作用于毛细胞和螺旋神经节细胞,反馈抑制耳蜗的反应性,防止谷氨酸过量释放引起的兴奋性毒性。CGRP被证实存在于耳蜗传出神经系统以及支配耳蜗血管的自主神经系统,是耳蜗传出神经系统众多递质中的一员,并且在离断的腓肠神经中CGRP可以密切损伤神经元与周围施万细胞的联系,促进轴突再生。在本实验研究中,我们旨在观察外源性CGRP对谷氨酸引起的螺旋神经节细胞损伤有无直接预防作用,并检测在螺旋神经节细胞有无CGRP受体的表达。方法：用出生3-5天的乳大鼠,分离、培养螺旋神经节细胞,以含10%优质胎牛血清的DMEM培养24 h,用500μM谷氨酸分别处理6、12、24 h提取细胞总蛋白以western blot检测caspase-3蛋白表达随时间的变化情况,另以1nM、10nM、100nM的CGRP预处理后加入500μM谷氨酸培养24 h提取细胞总蛋白检测caspase-3表达情况。正常培养24 h的螺旋神经节细胞提取细胞总RNA和总蛋白,应用RT-PCR及western blot方法检测CGRP I型受体组成蛋白成分的表达。结果： 1.谷氨酸钠500μM处理后6h螺旋神经节细胞内caspase-3蛋白表达量即开始增加,并持续到应用后12 h,处理后24 h表达量下降。 2.应用1nMCGRP预处理后与单独应用500μM谷氨酸钠损伤24h相比,螺旋神经节细胞中caspase-3表达量减少,然而随CGRP浓度的增加螺旋神经节细胞中caspase-3的表达亦开始上调。 3.RT-PCR及western-blot结果显示在螺旋神经节细胞无CGRP I型受体的表达。结论： 1.在暴露于高浓度谷氨酸初期即会引起螺旋神经节细胞内caspase-3蛋白的激活,导致部分神经元细胞凋亡。 2.CGRP作用具有双重效应：低浓度可以减少caspase-3的激活,有效地避免螺旋神经节细胞的凋亡；高浓度CGRP可引起剂量依赖的caspase-3激活水平的增加。 3.CGRP在螺旋神经节细胞发挥作用并非通过Ⅰ型受体途径。
[Abstract]:Spiral ganglion cells are the first stage neurons in the auditory conduction pathway. The stimulation of glutamate, an excitatory transmitter released by hair cells, produces excitatory potentials, which are transmitted to the ventral and dorsal cochlear nuclei at the junction of the medullary pontine. However, under some pathological conditions, such as noise, ischemia, hypoxia, infection and ototoxic drug use, glutamate accumulation can be induced, spiral ganglion cells damage and irreversible hearing loss can be caused by many ways. The efferent nerve system acts on the hair cells and the spiral ganglion cells through the nerve pathway, and feedback inhibits the response of the cochlea. CGRP is found to exist in the cochlear efferent system and the autonomic nervous system that innervates the cochlear vessels, and is one of the many transmitters in the cochlear efferent system. Moreover, CGRP in the transected sural nerve can damage the connection between neurons and peripheral Schwann cells and promote axon regeneration. In this study, we investigated the effects of exogenous CGRP on glutamate induced injury of spiral ganglion cells, and detected the expression of CGRP receptors in spiral ganglion cells. Methods: helical ganglion cells were isolated and cultured from 3 to 5 day old neonatal rats. DMEM containing 10% high quality fetal bovine serum was cultured for 24 h. 渭 M glutamate was used to extract the total cell protein for 24 h. Western blot was used to detect the change of caspase-3 protein expression over time, and 1nM 10nM was used to detect the expression of caspase-3 protein. After pretreatment with CGRP of 100nM, 500 渭 M glutamic acid was added to culture for 24 h. Total protein was extracted to detect the expression of caspase-3. The total RNA and total protein were extracted from spiral ganglion cells cultured for 24 h. The expression of CGRP type I receptor components was detected by RT-PCR and western blot methods. Results: 1. The expression of caspase-3 protein in spiral ganglion cells began to increase 6 h after treatment with 500 渭 M glutamate, and continued to decrease 24 h after treatment for 12 h. 2. After pretreatment with 1nMCGRP, the expression of caspase-3 in spiral ganglion cells decreased compared with 500 渭 M sodium glutamate alone for 24 hours. However, the expression of caspase-3 in spiral ganglion cells increased with the increase of CGRP concentration. 3.RT-PCR and western-blot showed that there was no expression of CGRP type I receptor in spiral ganglion cells. Conclusion: 1. The activation of caspase-3 protein in spiral ganglion cells at the early stage of exposure to high concentration of glutamate resulted in apoptosis of some neurons. The action of 2.CGRP has dual effects: low concentration can reduce the activation of caspase-3 and effectively avoid apoptosis of spiral ganglion cells, and high concentration of CGRP can increase the level of caspase-3 activation in a dose-dependent manner. The role of 3.CGRP in spiral ganglion cells is not via type I receptor pathway.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764.431

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