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  本文關(guān)鍵詞： 谷氨酸 內(nèi)毛細胞 纖毛 免疫熒光 耳蝸 谷氨酸 谷氨酰胺合成酶 谷氨酸-谷氨酰胺循環(huán)　出處：《華中科技大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分外源性谷氨酸對體外培養(yǎng)大鼠耳蝸毛細胞損傷的研究 目的:觀察體外培養(yǎng)的大鼠毛細胞暴露在外源性谷氨酸環(huán)境下的損傷情況。 方法:將出生3天的新生SD大鼠33只分Ⅰ～Ⅺ組,每組3只取基底膜,去掉頂回后培養(yǎng)24小時后,Ⅰ～Ⅵ組分別加入不同濃度的谷氨酸(0.1mM,0.5mM,1.0 mM,5.0 mM,10 mM,20 mM);Ⅶ～Ⅹ組分別在同一谷氨酸濃度(20mM)下作用6H,12H,24H, 72H;Ⅺ組為正常對照組。用免疫熒光染色的方法(phalloidin和DAPI染色)觀察毛細胞胞核和纖毛,每個樣本在顯微鏡下隨機取3個不同視野計數(shù)內(nèi)毛細胞纖毛之和,并進行統(tǒng)計分析。 結(jié)果:①培養(yǎng)24h后,Ⅰ組內(nèi)毛細胞沒有出現(xiàn)明顯損傷,纖毛排列整齊,無錯位、倒伏、缺失。Ⅱ～Ⅵ組,內(nèi)毛細胞均出現(xiàn)損傷,并伴有纖毛紊亂,倒伏,缺失。②在20倍的物鏡下觀察,Ⅶ～Ⅺ組內(nèi)毛細胞纖毛數(shù)分別為106.83±2.787、75.00±3.742、59.33±2.582、46.17±3.920、109.50±2.168,Ⅶ～Ⅹ組與正常對照組比較內(nèi)毛細胞纖毛數(shù)減少(p0.05),并且殘存的纖毛數(shù)與暴露在谷氨酸中的時間呈量效關(guān)系(p0.05);③在60倍的物鏡下觀察,20mM谷氨酸作用24小時后,基底膜中回內(nèi)毛細胞纖毛的殘存數(shù)(47.33±3.983)比底回內(nèi)毛細胞纖毛的殘存數(shù)(33.17±4.579)要多(p0.05)。 結(jié)論:①外源性谷氨酸對體外培養(yǎng)基底膜的內(nèi)毛細胞最小損傷濃度約為0.5mM;②體外培養(yǎng)的基底膜暴露在20mM谷氨酸環(huán)境中,內(nèi)毛細胞6H開始出現(xiàn)損傷,12H后有明顯纖毛缺失,并且隨著暴露在谷氨酸中的時間延長,細胞損傷越嚴重,纖毛缺失得越厲害;③暴露在相同濃度和相同時間的谷氨酸環(huán)境中,基底膜底回的內(nèi)毛細胞損傷和纖毛缺失要比中回的嚴重。 第二部分外源性谷氨酸對大鼠耳蝸谷氨酰胺合成酶表達的相關(guān)性研究 目的:探討大鼠耳蝸谷氨酰胺合成酶表達部位和導(dǎo)入外源性谷氨酸后其表達的變化。 方法:成年SD大鼠40只,分成Ⅰ~Ⅵ組,Ⅰ組10只為正常對照組,Ⅱ~Ⅵ組為手術(shù)組,每組6只。其中Ⅱ組導(dǎo)入外淋巴液,Ⅲ~Ⅵ組分別為20mM谷氨酸導(dǎo)入后12h,1天,3天,7天組。利用免疫組織化學(xué)、蛋白免疫印記法觀察大鼠耳蝸內(nèi)谷氨酰胺合成酶的分布和表達變化。 結(jié)果:谷氨酰胺合成酶在耳蝸Corti器內(nèi)支持細胞,螺旋神經(jīng)節(jié)周圍膠質(zhì)細胞和骨螺旋板內(nèi)均有表達;導(dǎo)入谷氨酸后1天組谷氨酰胺合成酶表達增多(p0.05),對照組,12h、3天、7天組與正常組表達無差異(p0.05)。 結(jié)論:谷氨酰胺合成酶在耳蝸Corti器內(nèi)支持細胞內(nèi)也有表達,與谷氨酸-谷氨酰胺循環(huán)假說相符;外源性谷氨酸干預(yù)后,谷氨酰胺合成酶表達會上調(diào),可能是機體的代償保護作用。
[Abstract]:The first part of the study of exogenous glutamic acid on rat cochlear hair cell injury in vitro
Objective: To observe the damage of rat hair cells exposed to exogenous glutamic acid in vitro.
Methods: SD rats born 3 days only 33 points I ~ - group, 3 rats in each group from the basement membrane, removing the top back after cultured for 24 hours, I ~ VI groups were glutamic acid with different concentrations (0.1mM, 0.5mM, 1 mM, 5 mM, 10 mM, 20 mM); VII ~ x group were in the same concentration of glutamic acid (20mM) under the action of 6H, 12H, 24H, 72H; Xi group was the normal control group. Using immunofluorescence staining method (phalloidin and DAPI staining) on hair cell nuclei and cilia, each sample under the microscope were randomly selected from 3 different fields of vision counting inner hair cell the cilia, and statistical analysis.
Results: after 24h, I group of inner hair cells did not appear obvious damage, cilia arranged neatly, no dislocation, lodging, missing. II - VI group, inner hair cells are damaged, and accompanied by cilia disorder. There were lack of lodging in the objective 20 times, belong to hair cell cilia number Xi in the group were 106.83 + 2.787,75.00 + 3.742,59.33 + 2.582,46.17 + 3.920109.50 + 2.168, VII ~ x group compared with the control group to reduce the number of inner hair cell cilia (P0.05), and the remaining number of cilia with exposure to glutamate in time and dose effect relationship (P0.05); to observe in the lens at 60 times 24 hours after 20mM, the role of glutamate, residual number of basement membrane in inner hair cell cilia (47.33 + 3.983) than the remaining number of bottom back inner hair cell cilia (33.17 + 4.579) to (P0.05).
Conclusion: the smallest injury concentration of inner hair cells of exogenous glutamate on the cultured basilar membrane is about 0.5mM; the basement membrane in vitro exposure to glutamate in the 20mM environment, the inner hair cells began to appear 6H injury, 12H had obvious cilia missing, and with the extension of exposure in glutamic acid in time, cell damage more seriously, the more severe the loss of cilia; exposure at the same concentration and the same time the glutamic acid environment, basement membrane bottom back inner hair cell damage and loss of cilia than in the back.
Study on the correlation of glutamine synthetase expression in rat cochlea by exogenous glutamic acid in the second part
Objective: To investigate the expression of glutamine synthetase in the rat cochlea and the expression of glutamic acid after introducing exogenous glutamic acid.
Methods: 40 adult SD rats, divided into 1 ~ VI groups, group I 10 as normal control group, group II to VI for the surgical group, 6 rats in each group. The group into the perilymph, III ~ VI group were 20mM into 12h after glutamate for 1 days, 3 days, 7 days group using immunohistochemistry, Western blot method to observe the change of the distribution of the rat cochlea and the expression of glutamine synthetase.
Results: glutamine synthetase supporting cells in the organ of Corti, the expression of glial cells and spiral ganglion spiral plate has introduced 1 days after the group; glutamate glutamine synthetase expression increased (P0.05), the control group, 12h, for 3 days, there is no difference between the 7 day group and normal group expression (P0.05).
Conclusion: glutamine synthetase is also expressed in the supporting cells of cochlear Corti, which is consistent with the glutamate cycle hypothesis. Glutamine synthetase expression is upregulated after exogenous glutamate, which may be the compensatory protective effect of the body.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R764

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本文編號：1463997