水杨酸钠作用后大鼠耳蜗热休克蛋白27表达的研究

目的　研究水杨酸钠作用后大鼠耳蜗热休克蛋白 2 7(heatshockprotein2 7,HSP2 7)的表达特点 ,探讨HSP2 7在水杨酸钠耳毒性中的作用及意义。方法　用免疫组化SP法结合图像分析技术检测水杨酸钠注射后大鼠耳蜗HSP2 7的表达。结果　HSP2 7在正常及水杨酸钠作用后大鼠耳蜗各回均有不同程度的染色 ,主要阳性部位是Corti器、螺旋神经节、螺旋韧带和血管纹。但水杨酸钠作用后HSP2 7在耳蜗各阳性部位的表达均明显增强 ,其中以Corti器的表达更明显 ,P值均小于 0 .0 1。结论　HSP2 7在大鼠耳蜗组织表达有选择性 ,水杨酸钠能够明显诱导HSP2 7在大鼠耳蜗中的表达 ,HSP2 7可能对耳蜗形态结构损害后的修复起作用 ,且与耳鸣的产生发展可能有关。     

水杨酸钠作用后大鼠耳蜗K-Cl协同转运体家族mRNA表达研究

目的 研究水杨酸钠作用后大鼠耳蜗K-Cl协同转运体家族(K - Cl- cotransporter ,KCC)成员KCC1～4mRNA的表达情况,探讨KCC在水杨酸盐耳毒性中的作用及意义.方法 选用30只正常大鼠,随机分成五组,每组6只:第一组为预实验组,未给予任何处理,采用逆转录PCR技术检测大鼠耳蜗中KCC1～4mRNA表达情况;第二、三、四、五组分别为对照组(不用药)、慢性组(肌肉注射水杨酸钠175 mg/kg,2次/天,连用14天后处死观察)、急性组(一次肌肉注射水杨酸钠400 mg/kg后处死观察)、恢复组(水杨酸钠用量及用法同慢性组,停药14天后处死观察),运用荧光定量PCR技术比较各组大鼠耳蜗内KCC1～4mRNA表达情况. 结果 KCC1～4mRNA在正常成年大鼠耳蜗中均有表达;在对照组及水杨酸钠作用后大鼠耳蜗KCC1～4mRNA表达均有变化,急性组KCC1mRNA、慢性组和恢复组KCC2mRNA、恢复组KCC4mRNA表达水平高于对照组,差异有统计学意义(P<0.05),急性组KCC2mRNA、急性组、慢性组和恢复组KCC3mRNA以及急性组、慢性组KCC4mRNA表达水平低于对照组,差异有统计学意义(P<0.05). 结论 KCC1～4mRNA在正常大鼠耳蜗中均有表达,提示其可能共同参与耳蜗内淋巴液Cl-浓度的维持;在水杨酸钠作用后KCC1～4mRNA表达的不同变化,破坏了耳蜗内淋巴液Cl-浓度平衡,影响外毛细胞能动性.     

热休克蛋白27在耳蜗基底膜上的表达及其意义

目的探讨热休克蛋白27(HSP27)在耳蜗基底膜上的分布及意义。方法应用免疫荧光标记技术和激光扫描共聚焦显微镜技术(laserscanningconfocalmicroscopy,LSCM)检测HSP27在正常成年大鼠耳蜗基底膜上的表达。结果HSP27主要表达于耳蜗基底膜Corti器中外毛细胞的表皮板及其侧壁、内外柱细胞表面、外柱细胞的头、体及其足板,而内毛细胞及其它支持细胞上未见其表达。结论在发育成熟的正常耳蜗中,HSP27主要是通过稳定肌动蛋白微丝的正常结构,维持细胞骨架的稳定,维持耳蜗内活性氧在正常水平及执行分子伴侣的功能,保护细胞,从而达到维持耳蜗正常生理功能的作用。     

水杨酸钠对大鼠耳蜗NKCC1 mRNA表达的影响

目的研究水杨酸钠作用后大鼠耳蜗Na^＋-K^＋-2Cl^-联合转运体（Na^＋-K^＋-2Cl^-co-trans-porter,NKCC1）mRNA的表达变化情况,探讨NKCC1在水杨酸钠肌注后引起大鼠耳蜗主动性改变中的作用和意义。方法选用24只正常成年SD大鼠,随机分成四组,每组6只：正常对照组（无特殊处理）、急性组（一次肌注水杨酸钠400mg／kg后处死）、慢性组（肌注水杨酸钠175mg／kg,2次／天,连续用药14天后处死）,恢复组（给药方法及时间同慢性组,停药14天后处死）,运用荧光定量PCR技术比较四组大鼠耳蜗NKCC1mRNA的表达变化。结果NKCC1mRNA在正常对照组、急性组、慢性组、恢复组均有表达,慢性组、恢复组的表达量均高于正常组,差异均有统计学意义（P〈0．05）;急性组NKCC1mRNA的表达低于正常组,差异有统计学意义（P〈0．05）;恢复组NKCC,mRNA的表达低于慢性组,差异有统计学意义（P〈0．05）。结论NKCC1mRNA在正常大鼠中有表达,提示其对维持内淋巴液中Cl^-的平衡有一定作用;水杨酸钠肌注可以引起大鼠耳蜗NKCC1mRNA表达变化,可能通过改变耳蜗内淋巴液Cl^-浓渡平衡,而影响外毛细胞的电能动性。     

补肾活血化痰开窍方对耳鸣大鼠耳蜗螺旋神经节神经元5-HT1B、2C受体表达的影响北大核心CSCD

目的探讨补肾活血化痰开窍方对耳鸣大鼠耳蜗螺旋神经节神经元(spiral ganglion neurons,SGN)中5羟色胺(serotonin,5-HT)1B、2C受体的表达及意义。方法 60只健康SPF级雄性大鼠随机分为正常组10只(生理盐水组),耳鸣模型组50只(采用水杨酸钠腹腔注射联合"饮水抑制"大鼠行为学实验方法建立大鼠耳鸣模型),耳鸣模型组造模成功后再分为水杨酸钠组10只、中药组30只(补肾活血化痰开窍方低、中、高剂量组各10只)、西药组(卡马西平组)10只,除水杨酸钠组给予生理盐水灌胃外,各组给予相应药物干预8周,造模成功后运用免疫组化法检测各组耳蜗SGN中5-HT1B、2C受体的表达。结果耳鸣造模成功并给予相应药物干预8周后,各组大鼠耳蜗SGN中均有5-HT1B、2C受体的表达,与生理盐水组相比,水杨酸钠组中5-HT1B表达减少,5-HT2C表达明显增多;与水杨酸钠组相比,中药各剂量组耳蜗SGN中5-HT1B表达增多,2C表达均明显降低。结论耳鸣大鼠耳蜗SGN中的5-HT1B受体表达减少、5-HT2C表达增加,应用补肾活血化痰开窍方后5-HT1B表达增多而5H2C表达降低,产生了拮抗作用,该方可能应用于耳鸣的治疗。     

热休克蛋白60在药物性聋大鼠模型中的表达变化

目的探讨HSP60在大鼠耳蜗中的表达及硫酸卡那霉素损伤后的表达变化。方法正常成年雌性SD大鼠20只随机分为两组:对照组(生理盐水)和实验组(硫酸卡纳霉素),按500mg/kg剂量每天给药一次,连续腹腔注射21天。ABR检测听力变化;实时定量PCR和Western Blot分别检测HSP60的m RNA和蛋白表达量的变化;免疫荧光染色观察HSP60在耳蜗中的表达变化及分布。结果 ABR检测显示实验组较对照组明显升高(P<0.05)。实时定量PCR、WesternBlot和基底膜铺片免疫荧光染色的结果显示实验组HSP60表达量降低(P<0.05)。冰冻切片免疫荧光染色,观察到HSP60表达于Corti器上的支持细胞内。结论 HSP60表达于支持细胞,正常生理情况下即表达,慢性药物中毒性耳聋模型中表达量降低,推测HSP60可能参与了药物性耳聋的发生过程。     

正常及慢性水杨酸钠作用后大鼠耳蜗总RNA的提取和产量

目的 探讨提取正常及慢性水杨酸钠作用的大鼠耳蜗总RNA的方法并测算其产量，为进一步研究提供实验数据。方法 22只健康Wistar大鼠随机分为两组，实验组12只进行2周的水杨酸钠肌肉注射，对照组10只不作处理。联用TRIzol和RNeary法分别抽取两组大鼠耳蜗的总RNA，用分光光度法和电泳测总RNA的产量和质量。结果 l0只正常组大鼠耳蜗得到总RNA7．82μg，12只长期水杨酸钠作用后的大鼠耳蜗得到总RNA4．82μg，两组平均产量不同，A260／A280值分别为2．05和2．08，提示RNA纯度高，电泳结果提示总RNA无降解。结论 两种方法合用提取总RNA的质量高，可满足后续实验要求。长期水杨酸钠作用后大鼠的耳蜗总RNA较正常大鼠耳蜗总RNA产量上有改变，进一步的研究可利用基因表达谱技术研究具体基因变化。     

Differential gene expression profiles in salicylate ototoxicity of the mouse

CONCLUSION: This study demonstrated differential gene expression profiles in salicylate ototoxicity with oligonucleotide microarray. This study may also provide basic information on candidate genes associated with hearing loss and/or tinnitus or recovery after salicylate-induced cochlear dysfunction. OBJECTIVES: Salicylate ototoxicity is accompanied by temporary hearing loss and tinnitus. The purpose of the present study was to evaluate the gene expression profiles in the mouse cochlea with salicylate ototoxicity using DNA microarray. MATERIALS AND METHODS: The subject mice were injected intraperitoneally with 400 mg/kg of sodium salicylate; an approximate 30 dB threshold shift that was observed by auditory brainstem response was achieved 3 h after an injection of sodium salicylate and the hearing threshold returned to within normal range at 3 days. Differential gene expression profiles at 3 h after salicylate injection in comparison to the normal cochlea were analyzed with DNA microarray technology. RESULTS: No ultrastructural changes in the mice cochlea were observed by TEM at 3 h after salicylate injection. Microarray revealed that 87 genes were up-regulated twofold or more in the mouse cochlea with salicylate ototoxicity in comparison to the normal cochlea. Among these genes, increased expression levels of 30 functional genes were confirmed by semi-quantitative RT-PCR.     

Differential gene expression profiles in salicylate ototoxicity of the mouse

Conclusion. This study demonstrated differential gene expression profiles in salicylate ototoxicity with oligonucleotide microarray. This study may also provide basic information on candidate genes associated with hearing loss and/or tinnitus or recovery after salicylate-induced cochlear dysfunction. Objectives: Salicylate ototoxicity is accompanied by temporary hearing loss and tinnitus. The purpose of the present study was to evaluate the gene expression profiles in the mouse cochlea with salicylate ototoxicity using DNA microarray. Materials and methods: The subject mice were injected intraperitoneally with 400 mg/kg of sodium salicylate; an approximate 30 dB threshold shift that was observed by auditory brainstem response was achieved 3 h after an injection of sodium salicylate and the hearing threshold returned to within normal range at 3 days. Differential gene expression profiles at 3 h after salicylate injection in comparison to the normal cochlea were analyzed with DNA microarray technology. Results: No ultrastructural changes in the mice cochlea were observed by TEM at 3 h after salicylate injection. Microarray revealed that 87 genes were up-regulated twofold or more in the mouse cochlea with salicylate ototoxicity in comparison to the normal cochlea. Among these genes, increased expression levels of 30 functional genes were confirmed by semi-quantitative RT-PCR.     

急慢性水杨酸钠注射后大鼠耳蜗基因表达谱研究

目的运用基因芯片技术和自组织映射(self-organizing map，SOM)聚类分析，在基因水平研究急慢性水杨酸钠注射后大鼠耳蜗电生理不同变化机制。方法取急性、慢性水杨酸钠注射的大鼠和正常大鼠，断头处死，剥离耳蜗，在显微镜下去掉骨质外壳，将其内容物匀浆，抽提总RNA，纯化后的总RNA逆转录成cDNA，体外转录成cRNA杂交探针，探针先与测试芯片杂交，确定无问题后再与含有8800多个基因的寡核苷酸基因芯片杂交，得到3种情况的耳蜗基因表达谱，对有变化的基因进行SOM聚类分析，与不同电生理变化进行类比，最后利用标准基因词汇体系对类比结果中的基因进行电子注释。采用实时定量逆转录多聚酶链反应验证芯片结果。结果样品探针质量佳，芯片实验结果可信，定量逆转录多聚酶链反应与基因芯片结果基本相符。聚类分析得到6类不同表达特性的基因，其中聚类3和4符合类比条件，基因词汇体系分析分别得到46和30个基因，涉及细胞信号传递、细胞活动、代谢、免疫反应和神经髓鞘形成等，可能与电生理变化有关。结论运用基因芯片技术和SOM聚类分析研究水杨酸钠注射引起电生理变化相关基因，可能为电生理变化机制提供新的思路。     

Differential protein expression profiles in salicylate ototoxicity of the mouse cochlea

The purpose of this study was to investigate protein expression profiles of salicylate ototoxicity using proteomic analysis, and to identify whether salicylates induce apoptosis in organotypic culture of mouse cochlear cells. The adult mice were injected intraperitoneally with 400 mg/kg of sodium salicylate. Approximately 30 dB threshold shift was observed 3 h after the injection, and the hearing threshold returned to normal range within 3 days. Proteomic analysis of mouse cochlea was performed 3 h after salicylate injection, because this was the time to show maximal ototoxic effect in salicylate intoxication. Expression pattern of proteomic analysis at 3 h was compared with those of normal cochlea and cochlea 3 days after salicylate injection. Sixteen proteins were transiently up-regulated threefolds or more at 3 h after the injection compared with normal cochlea, and three proteins were down-regulated at 3 h. Similar protein expression profiles were also observed between normal and 3 days group. These up-regulated and down-regulated proteins at 3 h were analyzed by MALDI-TOF MS. The mRNA expressions of nine selected genes from 16 up-regulated protein profiles were also investigated by RT-PCR, and their expression levels at 3 h were found to be higher than those of normal cochlea. We also confirmed the ototoxicity of salicylate in organotypic culture of cochlear cells using MTT assay, Hoechst staining and DNA laddering assay in vitro, and found that salicylate decreased the viability of cells in a time and dose-dependent manner, and that induced apoptosis in organotypic culture of cochlear cells. This study demonstrated that some proteins can be related to salicylate ototoxicity, and provides basic information about candidate proteins which are related to pathologic changes in salicylate-induced ototoxicity.     

水杨酸钠中耳灌注对庆大霉素耳毒性的防护作用

目的 观察水杨酸钠经中耳局部灌注给药对庆大霉素耳毒性的防护作用。方法 24只健康杂色豚鼠均接受圆窗置管术，然后随机分成3组：Ⅰ组为生理盐水对照组；Ⅱ组为庆大霉素组，腹腔注射庆大霉素，经听泡灌注生理盐水；Ⅲ组为庆大霉素加水杨酸钠组，腹腔注射庆大霉素，经听泡灌注水杨酸钠。观察各组给药前后听性脑干反应阈的变化和给药后4周毛细胞损失情况。结果 听泡置管术后ABR反应阈无明显改变；给药后2周和4周，庆大霉素组ABR反应阈较庆大霉素加水杨酸组显著增高（P〈0．01），对照组ABR反应阈无显著变化。耳蜗铺片、毛细胞计数显示庆大霉素组外毛细胞严重缺失，以底回最明显，庆大霉素加水杨酸钠组外毛细胞损失较庆大霉素组轻（P〈0．05）。对照组无明显外毛细胞缺失。结论 水杨酸钠经中耳局部给药途径可减轻庆大霉素所致听功能损害和毛细胞缺失，可在一定程度上有效预防庆大霉素的耳毒性。     

长期注射水杨酸钠对大鼠下丘GAD67和GABAAα1、c-fos表达的影响

目的 通过观察长期注射水杨酸钠对大鼠听性脑干反应(ABR)及下丘GAD67和GABAAα1、c-fos表达的影响,探讨大鼠下丘GAD67、GABAAα1、c-fos表达的改变在水杨酸钠耳毒性中的可能机制。 方法 将24只成年健康Wistar大鼠随机分为2组:水杨酸钠组(肌肉注射10%水杨酸钠175 mg/kg,2次/d,连续28 d)、对照组(每天相同时间注射等量生理盐水,连续28 d)。两组大鼠在处死前进行ABR检测并观察ABR反应阈及波Ⅲ潜伏期的变化,然后将大鼠断头处死并迅速剥离下丘,采用实时荧光定量PCR(real-time PCR)、蛋白质印迹(Western blot)方法检测下丘GAD67、GABAAα1、c-fos mRNA及蛋白表达的变化。 结果 ①药物注射28 d后,水杨酸钠组较注射前以及对照组ABR反应阈明显升高,波Ⅲ潜伏期明显延长,差异有统计学意义(P<0.01);②水杨酸钠组GAD67、GABAAα1、c-fos mRNA及其蛋白表达水平较对照组明显升高,差异有统计学意义(P<0.01)。 结论 GAD67、GABAAα1、c-fos均不同程度参与了水杨酸钠耳毒性的发生发展过程, c-fos表达的上调则可能与水杨酸钠作用于机体后引起听觉中枢神经活动增强有关。     

Celiac disease and sensorineural hearing loss in children

Cisplatin ototoxicity is known to involve mainly the organ of Corti. Outer hair cells (OHCs), especially in the basal turn, are preferentially involved. One possible mechanism of ototoxicity might be alteration of the antioxidant system causing an increase in free radicals. It has been demonstrated that heat shock proteins (HSPs), which are believed to protect cells by dissolving and refolding misfolded or denatured protein are induced by various form of stress. HSP is also demonstrated to be induced by free radicals. The purpose of this study was to evaluate HSP 72 induction in cochlea following cisplatin injection in the animal model. Sprague-Dawley (SD) rats were injected intraperitoneally with normal saline as control or cisplatin at a dose of 5, 10 or 20 mg/kg. Cochleae were harvested 1, 3, 6 and 12 h after injection and compared with those of controls. Immunocytochemical study with surface preparation and Western blotting were performed to investigate the expression of HSP 72. Auditory brainstem response (ABR) was also recorded to assess functional change according to the dosage of cisplatin and duration after injection. In the 5 and 10 mg/kg groups, immunostaining for HSP 72 in the OHCs reached a plateau level at 3 h, which was maintained until 12 h after injection. The amount of immunoreactive OHCs in the 20 mg/kg group was smaller than those in 5 and 10 mg/kg groups and declined after 6 h. The bands for HSP 72 became less intense as the cisplatin dosage increased from 5 to 10 and 20 mg/kg in Western blotting. The change in ABR threshold was small in the 5 and 10 mg/kg groups and a marked change in threshold was observed in the 20 mg/kg group. Detection of HSP 72 after cisplatin injection could confirm the OHCs as one of the major injured cells in the cochlea. With a lethal dosage of cisplatin (20 mg/kg), HSP 72 expression was less prominent and declined after 6 h.