【摘要】： 螺旋神經(jīng)節(jié)細(xì)胞是聽覺傳導(dǎo)通路的第一級(jí)神經(jīng)元,其接受毛細(xì)胞釋放的興奮性遞質(zhì)谷氨酸的刺激產(chǎn)生興奮性電位,傳導(dǎo)至延髓腦橋連接處的蝸神經(jīng)腹核和背核。但在某些病理情況下,如：噪音、缺血、缺氧、感染、耳毒性藥物使用等,均可以引起谷氨酸的蓄積,通過多種途徑造成螺旋神經(jīng)節(jié)細(xì)胞的損傷,導(dǎo)致不可逆的聽力損傷。傳出神經(jīng)系統(tǒng)通過神經(jīng)通路作用于毛細(xì)胞和螺旋神經(jīng)節(jié)細(xì)胞,反饋抑制耳蝸的反應(yīng)性,防止谷氨酸過量釋放引起的興奮性毒性。CGRP被證實(shí)存在于耳蝸傳出神經(jīng)系統(tǒng)以及支配耳蝸血管的自主神經(jīng)系統(tǒng),是耳蝸傳出神經(jīng)系統(tǒng)眾多遞質(zhì)中的一員,并且在離斷的腓腸神經(jīng)中CGRP可以密切損傷神經(jīng)元與周圍施萬細(xì)胞的聯(lián)系,促進(jìn)軸突再生。在本實(shí)驗(yàn)研究中,我們旨在觀察外源性CGRP對(duì)谷氨酸引起的螺旋神經(jīng)節(jié)細(xì)胞損傷有無直接預(yù)防作用,并檢測(cè)在螺旋神經(jīng)節(jié)細(xì)胞有無CGRP受體的表達(dá)。 方法： 用出生3-5天的乳大鼠,分離、培養(yǎng)螺旋神經(jīng)節(jié)細(xì)胞,以含10%優(yōu)質(zhì)胎牛血清的DMEM培養(yǎng)24 h,用500μM谷氨酸分別處理6、12、24 h提取細(xì)胞總蛋白以western blot檢測(cè)caspase-3蛋白表達(dá)隨時(shí)間的變化情況,另以1nM、10nM、100nM的CGRP預(yù)處理后加入500μM谷氨酸培養(yǎng)24 h提取細(xì)胞總蛋白檢測(cè)caspase-3表達(dá)情況。正常培養(yǎng)24 h的螺旋神經(jīng)節(jié)細(xì)胞提取細(xì)胞總RNA和總蛋白,應(yīng)用RT-PCR及western blot方法檢測(cè)CGRP I型受體組成蛋白成分的表達(dá)。 結(jié)果： 1.谷氨酸鈉500μM處理后6h螺旋神經(jīng)節(jié)細(xì)胞內(nèi)caspase-3蛋白表達(dá)量即開始增加,并持續(xù)到應(yīng)用后12 h,處理后24 h表達(dá)量下降。 2.應(yīng)用1nMCGRP預(yù)處理后與單獨(dú)應(yīng)用500μM谷氨酸鈉損傷24h相比,螺旋神經(jīng)節(jié)細(xì)胞中caspase-3表達(dá)量減少,然而隨CGRP濃度的增加螺旋神經(jīng)節(jié)細(xì)胞中caspase-3的表達(dá)亦開始上調(diào)。 3.RT-PCR及western-blot結(jié)果顯示在螺旋神經(jīng)節(jié)細(xì)胞無CGRP I型受體的表達(dá)。 結(jié)論： 1.在暴露于高濃度谷氨酸初期即會(huì)引起螺旋神經(jīng)節(jié)細(xì)胞內(nèi)caspase-3蛋白的激活,導(dǎo)致部分神經(jīng)元細(xì)胞凋亡。 2.CGRP作用具有雙重效應(yīng)：低濃度可以減少caspase-3的激活,有效地避免螺旋神經(jīng)節(jié)細(xì)胞的凋亡；高濃度CGRP可引起劑量依賴的caspase-3激活水平的增加。 3.CGRP在螺旋神經(jīng)節(jié)細(xì)胞發(fā)揮作用并非通過Ⅰ型受體途徑。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R764.431

【共引文獻(xiàn)】

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