电针耳穴对硫酸卡那霉素慢性致聋豚鼠听觉中枢蛋白质组的影响

发布时间：2018-01-04 11:04

本文关键词：电针耳穴对硫酸卡那霉素慢性致聋豚鼠听觉中枢蛋白质组的影响　出处：《西南医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:探讨硫酸卡那霉素慢性致聋后豚鼠听皮层蛋白质组的变化、电针耳穴对听皮层蛋白质组的影响及机制,以及抑制素和吞蛋白-A1在硫酸卡那霉素慢性致聋发生和发展过程中的作用及电针对其表达的影响和机制。方法:1、动物分组:耳廓反射正常、无耳毒性药物史成年杂色豚鼠105只,随机分为3组:对照组7只,硫酸卡那霉素模型组49只,硫酸卡那霉素+电针组49只。对照组:颈背部皮下注射生理盐水(500mg/kg.day),每日一次,连续注射7天;硫酸卡那霉素模型组:颈背部皮下注射硫酸卡那霉素(500mg/kg.day),每日一次,连续注射7天;根据标本采集时间不同,又分为第1、7、14、28、56、70和140天7组。硫酸卡那霉素+电针组:颈背部皮下注射硫酸卡那霉素(500mg/kg.day),每只豚鼠注射结束30min后予以电针针刺翳风、听宫穴,每次15min,每日一次,连续治疗7天;根据电针后标本采集的时间不同,也分为第1、7、14、28、56、70和140天7组。2、听皮层蛋白质组图谱的建立及差异蛋白的选取及鉴定:对照组、硫酸卡那霉素模型1-140天7组、硫酸卡那霉素+电针1-140天7组分别提取听皮层蛋白后,用Bradford法测定蛋白质浓度,分组进行蛋白质双向电泳。电泳后进行考染,拍照后结合PDquest软件分析,筛选并切取其中21个表达差异明显的蛋白点,应用质谱技术进行检测,通过胶内酶解、抽提酶解肽段、Zip Tip脱盐、MALDI-TOF/TOF质谱测试、软件分析数据来鉴定蛋白质。3、听皮层及下丘抑制素和吞蛋白-A1表达的研究:采用蛋白质印迹方法测定对照组、硫酸卡那霉素模型1-140天7组、硫酸卡那霉素+电针1-140天7组豚鼠听皮层及下丘抑制素和吞蛋白-A1的表达。4、统计学方法:用SPSS20.0软件进行统计学分析,蛋白表达灰度值结果以平均值±标准差(x±s)表示,采用方差分析,P0.05认为差异具有统计学意义,t检验比较组间差异。结果:1.灰度差异2倍以上差异表达蛋白斑点鉴定出的蛋白质分别是14-3-3蛋白、海马钙结合蛋白样蛋白1、视锥蛋白样蛋白1、γ-可溶性NSF附着蛋白、78k Da葡萄糖调节蛋白、ATP合酶α亚基、微管不稳定蛋白、角蛋白Ⅱ型细胞骨架1、钙结合蛋白、吞蛋白-A1、磷酸吡哆醛磷酸酶、血影蛋白、异质核核糖核蛋白C、肌酸激酶B型、角蛋白Ⅰ型细胞骨架10、热休克蛋白70同源物-3,抑制素和ZBTB32蛋白等18种蛋白质。2.听皮层组织中抑制素、吞蛋白-A1表达的变化:对照组听皮层抑制素表达的相对灰度为1.11±0.13,模型组的第1、7、14、28、56、70和140天的相对灰度分别为1.05±0.14、1.01±0.10、0.92±0.09、0.88±0.16、0.85±0.14、0.94±0.15、1.08±0.13;电针组第1、7、14、28、56、70和140天的表达分别为0.82±0.11、1.32±0.14、0.97±0.13、1.08±0.12、1.01±0.11、1.20±0.16、1.09±0.07。与对照组相比,模型组抑制素第14、28、56天表达降低(P0.05);与模型组比较,电针组第7、70天抑制素表达增高(P0.05)。对照组听皮层吞蛋白-A1的表达为0.42±0.10,模型组的第1、7、14、28、56、70和140天的表达分别为0.64±0.02、0.44±0.04、0.58±0.10、52±0.03、0.45±0.04、0.42±0.02和0.42±0.04;电针组第1、7、14、28、56、70和140天的表达分别为0.56±0.06、0.47±0.04、0.46±0.02、0.42±0.06、0.35±0.02、0.43±0.06和0.43±0.06。与对照组相比,模型组第1、14天吞蛋白-A1表达增高(P0.05),与模型组比较,电针组中的第14、28、56天吞蛋白-A1表达降低(P0.05)。方差分析硫酸卡那霉素对豚鼠听皮层抑制素表达的影响,F=2.185,P=0.075;电针对慢性致聋豚鼠豚鼠听皮层抑制素表达的影响,F=7.490,P0.001。方差分析硫酸卡那霉素对豚鼠听皮层吞蛋白-A1表达的影响,F=12.823,P0.001;电针对慢性致聋豚鼠豚鼠听皮层吞蛋白-A1表达的影响,F=6.421,P=0.001。3.下丘组织中抑制素、吞蛋白-A1表达的变化:对照组下丘的抑制素表达为1.29±0.07,模型组的第1、7、14、28、56、70和140天的表达分别为0.92±0.08、1.02±0.14、0.78±0.10、0.81±0.07、0.76±0.08、0.98±0.08、1.33±0.10;电针组第1、7、14、28、56、70和140天的表达分别为1.02±0.09、1.30±0.13、0.79±0.08、1.17±0.14、1.42±0.13、1.04±0.17、1.33±0.10。与对照组相比,模型组抑制素第1、7、14、28、56、70天表达降低(P0.05);与模型组比较,电针组第7、28、56天抑制素表达增高(P0.05)。对照组下丘的吞蛋白-A1表达为0.95±0.05,模型组的第1、7、14、28、56、70和140天的表达分别为0.97±0.09、0.98±0.08、1.52±0.14、1.14±0.11、0.95±0.08、0.99±0.10、0.96±0.11;电针组第1、7、14、28、56、70和140天的表达分别为0.96±0.08、0.99±0.10、0.95±0.10、0.98±0.09、0.92±0.02、0.72±0.06、0.97±0.10。与对照组比较,模型组第14、28天表达增高(P0.05),与模型组比较,电针组第14、70天吞蛋白-A1表达降低(P0.05)。方差分析硫酸卡那霉素对豚鼠下丘抑制素表达的影响,F=17.210,P0.001;电针对慢性致聋豚鼠下丘抑制素表达的影响,F=12.544,P0.001。方差分析硫酸卡那霉素对豚鼠下丘吞蛋白-A1表达的影响,F=15.679,P0.001;电针对慢性致聋豚鼠下丘吞蛋白-A1表达的影响,F=5.476,P0.05。结论:1.14-3-3蛋白等18种蛋白质可能参与了硫酸卡那霉素慢性致聋的发生和发展过程。电针听宫、翳风穴可能通过调整以上蛋白质的表达来影响硫酸卡那霉素慢性致聋的发生和发展。2.抑制素可能通过调控线粒体功能、ROS的产生以及神经元对伤害的易感性,参与硫酸卡那霉素慢性致聋的过程。电针耳穴可能通过增加抑制素的表达保护线粒体功能,降低ROS产生,促进听皮层和下丘的功能重塑。3.吞蛋白-A1可能通过参与突触囊泡的回收过程以及一系列信号传导通路的调节,参与硫酸卡那霉素慢性致聋的过程。电针耳穴可能通过降低吞蛋白-A1的表达,抑制JNK等信号转导途径的激活,促进听皮层和下丘功能的恢复与重建。
[Abstract]:Objective: To investigate the chronic kanamycin induced deafness in guinea pigs after listening to the changes of cortical protein group, electroacupuncture effects on auditory cortex ear proteome and mechanism, and swallow inhibin and protein -A1 in chronic kanamycin induced deafness and electroacupuncture effect and the development process of the influence on the expression and mechanism. Methods: 1 animal grouping: normal auricle reflex, no history of ototoxic drugs only 105 adult pigmented guinea pigs, were randomly divided into 3 groups: control group 7, kanamycin sulfate 49 rats in model group and kanamycin sulfate + EA group 49. Control group: subcutaneous injection of saline (500mg/kg.day), once a day, 7 days of continuous injection of kanamycin sulfate; model group: subcutaneous injection of kanamycin sulfate (500mg/kg.day), once a day, continuous injection of 7 days; according to the time of sample collection, and divided into 7 groups at 1,7,14,28,56,70 and 140 days. Kanamycin sulfate EA group: subcutaneous injection of kanamycin sulfate (500mg/kg.day), 30min after the injection of each guinea pig to acupuncture Yifeng, Tinggong, 15min each time, once a day for 7 consecutive days; according to the specimen collection time after acupuncture, is also divided into 7 sections 1,7,14,28,56,70 and 140 days group.2, listen to the selection and establishment and identification of differential proteins of cortex proteome map: control group, kanamycin sulfate 1-140 7 day model group, kanamycin sulfate + EA 1-140 day of the 7 groups were extracted from the auditory cortex protein, protein concentration was determined by Bradford method, two-dimensional electrophoresis of protein electrophoresis grouping. After staining, PDquest software analysis combined with photographs, screening and cut 21 of them expressed significantly different protein spots by mass spectrometry were detected by gel digestion, peptide enzymatic extraction, Zip Tip desalting, MALDI-TOF/TOF mass spectrometry test software Analysis of data to identify protein.3, listen to the expression of inhibin and swallow protein -A1 in cerebral cortex and hypothalamus: Determination of control group by Western blot method, kanamycin sulfate 1-140 7 day model group, the statistical method of kanamycin sulfate + EA 1-140 day 7 groups of guinea pigs to the expression of.4 in cortex and hypothalamus of inhibin and swallow the eggs white -A1: using SPSS20.0 software for statistical analysis, the protein expression in average and standard deviation and the gray value of (x + s) said, using analysis of variance, P0.05 considered statistically significant difference, the difference between the groups t test. Results: 1. gray difference more than 2 times the differentially expressed protein spots identified proteins respectively. Is the hippocampus 14-3-3 protein, calcium binding protein like protein 1, cone protein gamma like protein 1, soluble NSF attachment protein, Da glucose regulated protein 78k, ATP synthase alpha subunit, microtubule destabilizing protein, keratin type II cell skeleton 1, calcium Swallow binding protein, protein -A1, pyridoxal phosphate phosphatase, spectrin, heterogeneous nuclear ribonucleoprotein C, creatine kinase B, keratin type 10 cytoskeleton, heat shock protein 70 homolog -3, inhibin ZBTB32 protein and 18 proteins such as.2. to cortex expression of inhibin, swallow protein -A1: the control group to the relative gray cortex inhibin expression of is 1.11 + 0.13, relative gray days 1,7,14,28,56,70 and 140 model group were 1.05 + 0.14,1.01 + 0.10,0.92 + 0.09,0.88 + 0.16,0.85 + 0.14,0.94 + 0.15,1.08 + 0.13; Electroacupuncture group the expression of 1,7,14,28,56,70 and 140 days were 0.82 + 0.11,1.32 + 0.14,0.97 + 0.13,1.08 + 0.12,1.01 + 0.11,1.20 + 0.16,1.09 + 0.07. compared with the control group, model group inhibin day 14,28,56 expression decreased (P0.05); compared with the model group, EA group 7,70 days increased expression of inhibin (P0.05). The control group to listen to The expression of -A1 protein was 0.42 swallow cortex + 0.10, model group on days 1,7,14,28,56,70 and 140 expression were 0.64 + 0.02,0.44 + 0.04,0.58 + 0.10,52 + 0.03,0.45 + 0.04,0.42 + 0.02 and 0.42 + 0.04; the expression of 1,7,14,28,56,70 in electroacupuncture group and 140 days were 0.56 + 0.06,0.47 + 0.04,0.46 + 0.02,0.42 + 0.06,0.35 + 0.02,0.43 + 0.06 and 0.43 + 0.06. compared with the control group, the model group at 1,14 days to swallow the protein expression of -A1 increased (P0.05), compared with the model group, EA group in the first 14,28,56 days to swallow the decreased expression of -A1 protein (P0.05). Analysis of variance of kanamycin sulfate to influence on the expression of inhibin, cortex of guinea pig F=2.185, P=0.075 F=7.490; Electroacupuncture on chronic deafened guinea pigs in guinea pigs, cortex inhibin expression, P0.001. variance analysis of kanamycin sulfate to influence expression of -A1 protein in guinea pig cortex swallow F=12.823, P0.001; electric for chronic deafened guinea pigs Guinea pigs, -A1 F=6.421 protein expression in the cerebral cortex swallow, inhibin P=0.001.3. hypothalamic tissues, the expression of -A1 protein in control group: swallow the inferior colliculus was 1.29 + 0.07 inhibin expression, model group on days 1,7,14,28,56,70 and 140 expression were 0.92 + 0.08,1.02 + 0.14,0.78 + 0.10,0.81 + 0.07,0.76 + 0.08,0.98 + 0.08,1.33 + 0.10; the expression of 1,7,14,28,56,70 in electroacupuncture group and 140 days were 1.02 + 0.09,1.30 + 0.13,0.79 + 0.08,1.17 + 0.14,1.42 + 0.13,1.04 + 0.17,1.33 + 0.10. compared with the control group, model group, day 1,7,14,28,56,70 reduced expression of inhibin (P0.05); compared with the model group, EA group 7,28,56 days increased expression of inhibin (P0.05). The control group of inferior colliculus swallow the expression of -A1 protein was 0.95 + 0.05, model group on days 1,7,14,28,56,70 and 140 expression were 0.97 + 0.09,0.98 + 0.08,1.52 + 0.14,1.14 + 0.11,0.95 + 0.08,0.99 + 0.10,0. 96 + 0.11; the expression of 1,7,14,28,56,70 in electroacupuncture group and 140 days were 0.96 + 0.08,0.99 + 0.10,0.95 + 0.10,0.98 + 0.09,0.92 + 0.02,0.72 + 0.06,0.97 + 0.10. compared with the control group, model group, day 14,28 expression (P0.05), compared with the model group, EA group decreased at 14,70 days to swallow the protein expression of -A1 (P0.05) the analysis of variance. Effects of Kanamycin on expression of inhibin in guinea pig inferior colliculus F=17.210, P0.001; F=12.544 effect of Electroacupuncture on chronic, induced deafness in guinea pig inferior colliculus inhibin expression, P0.001. variance analysis of Kanamycin on the effect of protein -A1 expression in guinea pig inferior colliculus swallow F=15.679, P0.001; effect of electro acupuncture on chronic deafness. The expression of -A1 protein in guinea pig inferior colliculus swallow F=5.476 P0.05. conclusion: the expression of 1.14-3-3 18 protein may be involved in the chronic kanamycin induced deafness. The occurrence and development process of EA Tinggong, Yifeng acupoint may adjust the above The expression of the protein to influence chronic kanamycin induced deafness in the occurrence and development of inhibin.2. by regulating mitochondrial function and susceptibility to injury of neurons in ROS, in the process of chronic kanamycin induced deafness. Electroacupuncture at auricular points could increase the expression of anti mitochondrial function in the protection system, reduce the production of ROS listen to, promote functional remodeling.3. swallow protein -A1 cortex and inferior colliculus may be involved in synaptic vesicle recycling process and regulate a series of signal transduction pathways, involved in the process of chronic kanamycin induced deafness. Electroacupuncture at auricular points by reducing the expression of -A1 protein may swallow, inhibited the activation of JNK signal transduction pathway, promote listen to the restoration and reconstruction of the cortex and the hypothalamus function.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R245

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