外源性谷氨酸对大鼠耳蜗毛细胞损伤及谷氨酰胺合成酶表达的相关性研究

发布时间：2018-01-25 23:27

本文关键词： 谷氨酸内毛细胞纤毛免疫荧光耳蜗谷氨酸谷氨酰胺合成酶谷氨酸-谷氨酰胺循环　出处：《华中科技大学》2010年硕士论文　论文类型：学位论文

【摘要】：第一部分外源性谷氨酸对体外培养大鼠耳蜗毛细胞损伤的研究目的:观察体外培养的大鼠毛细胞暴露在外源性谷氨酸环境下的损伤情况。方法:将出生3天的新生SD大鼠33只分Ⅰ～Ⅺ组,每组3只取基底膜,去掉顶回后培养24小时后,Ⅰ～Ⅵ组分别加入不同浓度的谷氨酸(0.1mM,0.5mM,1.0 mM,5.0 mM,10 mM,20 mM);Ⅶ～Ⅹ组分别在同一谷氨酸浓度(20mM)下作用6H,12H,24H, 72H;Ⅺ组为正常对照组。用免疫荧光染色的方法(phalloidin和DAPI染色)观察毛细胞胞核和纤毛,每个样本在显微镜下随机取3个不同视野计数内毛细胞纤毛之和,并进行统计分析。结果:①培养24h后,Ⅰ组内毛细胞没有出现明显损伤,纤毛排列整齐,无错位、倒伏、缺失。Ⅱ～Ⅵ组,内毛细胞均出现损伤,并伴有纤毛紊乱,倒伏,缺失。②在20倍的物镜下观察,Ⅶ～Ⅺ组内毛细胞纤毛数分别为106.83±2.787、75.00±3.742、59.33±2.582、46.17±3.920、109.50±2.168,Ⅶ～Ⅹ组与正常对照组比较内毛细胞纤毛数减少(p0.05),并且残存的纤毛数与暴露在谷氨酸中的时间呈量效关系(p0.05);③在60倍的物镜下观察,20mM谷氨酸作用24小时后,基底膜中回内毛细胞纤毛的残存数(47.33±3.983)比底回内毛细胞纤毛的残存数(33.17±4.579)要多(p0.05)。结论:①外源性谷氨酸对体外培养基底膜的内毛细胞最小损伤浓度约为0.5mM;②体外培养的基底膜暴露在20mM谷氨酸环境中,内毛细胞6H开始出现损伤,12H后有明显纤毛缺失,并且随着暴露在谷氨酸中的时间延长,细胞损伤越严重,纤毛缺失得越厉害;③暴露在相同浓度和相同时间的谷氨酸环境中,基底膜底回的内毛细胞损伤和纤毛缺失要比中回的严重。第二部分外源性谷氨酸对大鼠耳蜗谷氨酰胺合成酶表达的相关性研究目的:探讨大鼠耳蜗谷氨酰胺合成酶表达部位和导入外源性谷氨酸后其表达的变化。方法:成年SD大鼠40只,分成Ⅰ~Ⅵ组,Ⅰ组10只为正常对照组,Ⅱ~Ⅵ组为手术组,每组6只。其中Ⅱ组导入外淋巴液,Ⅲ~Ⅵ组分别为20mM谷氨酸导入后12h,1天,3天,7天组。利用免疫组织化学、蛋白免疫印记法观察大鼠耳蜗内谷氨酰胺合成酶的分布和表达变化。结果:谷氨酰胺合成酶在耳蜗Corti器内支持细胞,螺旋神经节周围胶质细胞和骨螺旋板内均有表达;导入谷氨酸后1天组谷氨酰胺合成酶表达增多(p0.05),对照组,12h、3天、7天组与正常组表达无差异(p0.05)。结论:谷氨酰胺合成酶在耳蜗Corti器内支持细胞内也有表达,与谷氨酸-谷氨酰胺循环假说相符;外源性谷氨酸干预后,谷氨酰胺合成酶表达会上调,可能是机体的代偿保护作用。
[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.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764

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