本文關(guān)鍵詞：電針耳穴對(duì)硫酸卡那霉素慢性致聾豚鼠聽覺中樞蛋白質(zhì)組的影響　出處：《西南醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蛋白質(zhì)組 電針 硫酸卡那霉素 聽皮層 下丘 抑制素 吞蛋白-A1

【摘要】：目的:探討硫酸卡那霉素慢性致聾后豚鼠聽皮層蛋白質(zhì)組的變化、電針耳穴對(duì)聽皮層蛋白質(zhì)組的影響及機(jī)制,以及抑制素和吞蛋白-A1在硫酸卡那霉素慢性致聾發(fā)生和發(fā)展過(guò)程中的作用及電針對(duì)其表達(dá)的影響和機(jī)制。方法:1、動(dòng)物分組:耳廓反射正常、無(wú)耳毒性藥物史成年雜色豚鼠105只,隨機(jī)分為3組:對(duì)照組7只,硫酸卡那霉素模型組49只,硫酸卡那霉素+電針組49只。對(duì)照組:頸背部皮下注射生理鹽水(500mg/kg.day),每日一次,連續(xù)注射7天;硫酸卡那霉素模型組:頸背部皮下注射硫酸卡那霉素(500mg/kg.day),每日一次,連續(xù)注射7天;根據(jù)標(biāo)本采集時(shí)間不同,又分為第1、7、14、28、56、70和140天7組。硫酸卡那霉素+電針組:頸背部皮下注射硫酸卡那霉素(500mg/kg.day),每只豚鼠注射結(jié)束30min后予以電針針刺翳風(fēng)、聽宮穴,每次15min,每日一次,連續(xù)治療7天;根據(jù)電針后標(biāo)本采集的時(shí)間不同,也分為第1、7、14、28、56、70和140天7組。2、聽皮層蛋白質(zhì)組圖譜的建立及差異蛋白的選取及鑒定:對(duì)照組、硫酸卡那霉素模型1-140天7組、硫酸卡那霉素+電針1-140天7組分別提取聽皮層蛋白后,用Bradford法測(cè)定蛋白質(zhì)濃度,分組進(jìn)行蛋白質(zhì)雙向電泳。電泳后進(jìn)行考染,拍照后結(jié)合PDquest軟件分析,篩選并切取其中21個(gè)表達(dá)差異明顯的蛋白點(diǎn),應(yīng)用質(zhì)譜技術(shù)進(jìn)行檢測(cè),通過(guò)膠內(nèi)酶解、抽提酶解肽段、Zip Tip脫鹽、MALDI-TOF/TOF質(zhì)譜測(cè)試、軟件分析數(shù)據(jù)來(lái)鑒定蛋白質(zhì)。3、聽皮層及下丘抑制素和吞蛋白-A1表達(dá)的研究:采用蛋白質(zhì)印跡方法測(cè)定對(duì)照組、硫酸卡那霉素模型1-140天7組、硫酸卡那霉素+電針1-140天7組豚鼠聽皮層及下丘抑制素和吞蛋白-A1的表達(dá)。4、統(tǒng)計(jì)學(xué)方法:用SPSS20.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,蛋白表達(dá)灰度值結(jié)果以平均值±標(biāo)準(zhǔn)差(x±s)表示,采用方差分析,P0.05認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義,t檢驗(yàn)比較組間差異。結(jié)果:1.灰度差異2倍以上差異表達(dá)蛋白斑點(diǎn)鑒定出的蛋白質(zhì)分別是14-3-3蛋白、海馬鈣結(jié)合蛋白樣蛋白1、視錐蛋白樣蛋白1、γ-可溶性NSF附著蛋白、78k Da葡萄糖調(diào)節(jié)蛋白、ATP合酶α亞基、微管不穩(wěn)定蛋白、角蛋白Ⅱ型細(xì)胞骨架1、鈣結(jié)合蛋白、吞蛋白-A1、磷酸吡哆醛磷酸酶、血影蛋白、異質(zhì)核核糖核蛋白C、肌酸激酶B型、角蛋白Ⅰ型細(xì)胞骨架10、熱休克蛋白70同源物-3,抑制素和ZBTB32蛋白等18種蛋白質(zhì)。2.聽皮層組織中抑制素、吞蛋白-A1表達(dá)的變化:對(duì)照組聽皮層抑制素表達(dá)的相對(duì)灰度為1.11±0.13,模型組的第1、7、14、28、56、70和140天的相對(duì)灰度分別為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;電針組第1、7、14、28、56、70和140天的表達(dá)分別為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。與對(duì)照組相比,模型組抑制素第14、28、56天表達(dá)降低(P0.05);與模型組比較,電針組第7、70天抑制素表達(dá)增高(P0.05)。對(duì)照組聽皮層吞蛋白-A1的表達(dá)為0.42±0.10,模型組的第1、7、14、28、56、70和140天的表達(dá)分別為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;電針組第1、7、14、28、56、70和140天的表達(dá)分別為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。與對(duì)照組相比,模型組第1、14天吞蛋白-A1表達(dá)增高(P0.05),與模型組比較,電針組中的第14、28、56天吞蛋白-A1表達(dá)降低(P0.05)。方差分析硫酸卡那霉素對(duì)豚鼠聽皮層抑制素表達(dá)的影響,F=2.185,P=0.075;電針對(duì)慢性致聾豚鼠豚鼠聽皮層抑制素表達(dá)的影響,F=7.490,P0.001。方差分析硫酸卡那霉素對(duì)豚鼠聽皮層吞蛋白-A1表達(dá)的影響,F=12.823,P0.001;電針對(duì)慢性致聾豚鼠豚鼠聽皮層吞蛋白-A1表達(dá)的影響,F=6.421,P=0.001。3.下丘組織中抑制素、吞蛋白-A1表達(dá)的變化:對(duì)照組下丘的抑制素表達(dá)為1.29±0.07,模型組的第1、7、14、28、56、70和140天的表達(dá)分別為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;電針組第1、7、14、28、56、70和140天的表達(dá)分別為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。與對(duì)照組相比,模型組抑制素第1、7、14、28、56、70天表達(dá)降低(P0.05);與模型組比較,電針組第7、28、56天抑制素表達(dá)增高(P0.05)。對(duì)照組下丘的吞蛋白-A1表達(dá)為0.95±0.05,模型組的第1、7、14、28、56、70和140天的表達(dá)分別為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;電針組第1、7、14、28、56、70和140天的表達(dá)分別為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。與對(duì)照組比較,模型組第14、28天表達(dá)增高(P0.05),與模型組比較,電針組第14、70天吞蛋白-A1表達(dá)降低(P0.05)。方差分析硫酸卡那霉素對(duì)豚鼠下丘抑制素表達(dá)的影響,F=17.210,P0.001;電針對(duì)慢性致聾豚鼠下丘抑制素表達(dá)的影響,F=12.544,P0.001。方差分析硫酸卡那霉素對(duì)豚鼠下丘吞蛋白-A1表達(dá)的影響,F=15.679,P0.001;電針對(duì)慢性致聾豚鼠下丘吞蛋白-A1表達(dá)的影響,F=5.476,P0.05。結(jié)論:1.14-3-3蛋白等18種蛋白質(zhì)可能參與了硫酸卡那霉素慢性致聾的發(fā)生和發(fā)展過(guò)程。電針聽宮、翳風(fēng)穴可能通過(guò)調(diào)整以上蛋白質(zhì)的表達(dá)來(lái)影響硫酸卡那霉素慢性致聾的發(fā)生和發(fā)展。2.抑制素可能通過(guò)調(diào)控線粒體功能、ROS的產(chǎn)生以及神經(jīng)元對(duì)傷害的易感性,參與硫酸卡那霉素慢性致聾的過(guò)程。電針耳穴可能通過(guò)增加抑制素的表達(dá)保護(hù)線粒體功能,降低ROS產(chǎn)生,促進(jìn)聽皮層和下丘的功能重塑。3.吞蛋白-A1可能通過(guò)參與突觸囊泡的回收過(guò)程以及一系列信號(hào)傳導(dǎo)通路的調(diào)節(jié),參與硫酸卡那霉素慢性致聾的過(guò)程。電針耳穴可能通過(guò)降低吞蛋白-A1的表達(dá),抑制JNK等信號(hào)轉(zhuǎn)導(dǎo)途徑的激活,促進(jìn)聽皮層和下丘功能的恢復(fù)與重建。
[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.

【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R245

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