新生大鼠耳蜗血管纹缘细胞释放ATP的机制

目的 证实体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,并进一步探讨缘细胞释放ATP的机制.方法 分离、培养新生大鼠耳蜗血管纹缘细胞,采用生物发光法分别检测巴佛洛霉素A1、己二酸二癸酯( didecyl adipate,DDA)、细胞外K+、毒胡萝卜素、细胞外Ca2、U73122及马兜铃酸钠对细胞外液中缘细胞ATP释放的影响.结果 随着巴佛洛霉素A1浓度的增加,细胞外液中ATP的浓度明显下降;当DDA浓度增加时,细胞外液中ATP的浓度几乎呈线性增加.随着细胞外液中的K+浓度的增高,缘细胞释放的ATP浓度呈现上升趋势,当细胞外液中的K+浓度为9.15 mmol/L时,ATP的释放量达到峰值,之后随着K+浓度的继续升高ATP的释放量呈下降趋势.随着毒胡萝卜素浓度的增加,缘细胞释放的ATP浓度呈现明显下降的趋势.当细胞外Ca2+浓度为0 mmol/L时,缘细胞仍然释放ATP;Ca2+浓度增加与ATP的释放呈负相关,但当细胞外的Ca2+浓度达到1.25 mmol/L以上时,ATP的释放量维持在一个较稳定的水平.U73122的浓度在0.25～1.25 μmol/L时,其与缘细胞释放的ATP浓度呈负相关.当马兜铃酸钠的浓度为12.5 ～ 100.0 μmol/L,缘细胞ATP释放呈明显下降的趋势;当其浓度＞100.0 μmol/L时,ATP释放浓度趋于平稳.结论 体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,其释放量与钙泵、K+通道状态以及细胞内信号传导通路相关酶的活性有关.     

大鼠耳蜗中三磷酸腺苷表达及来源的初步研究

目的 研究大鼠耳蜗中三磷酸腺苷(adenosine triphosphate,ATP)的表达部位、来源及其可能的存在形式.方法 运用喹丫因染色技术,观察培养的大鼠耳蜗血管纹缘细胞、全膜迷路铺片和新鲜分离的单离外毛细胞;用免疫组化荧光染色法,观察突触素(synaptophysin,SYN)和小泡相关膜蛋白-2(vesicle-associated membrane protein-2/synaptobrevin,VAMP-2)在大鼠耳蜗中的表达.结果 培养的缘细胞胞浆中存在星点状喹丫因染色,全膜迷路铺片血管纹以外的区域和单离毛细胞中均未发现喹丫因的特异性染色.SYN和VAMP-2在大鼠耳蜗的内螺旋束、外螺旋束、Deiters细胞内侧缘和螺旋神经元(spiral ganglion neurons,SGNs)有共同表达.结论 大鼠耳蜗缘细胞胞浆中存在大量囊泡状的ATP,而毛细胞、支持细胞中的ATP可能以非囊泡的形式储存.内螺旋束、外螺旋束和SGNs中有可能存在SYN染色的ATP囊泡.     

新生大鼠耳蜗血管纹缘细胞中ATP存在的证据

目的:研究体外培养的新生大鼠耳蜗血管纹缘细胞中存在ATP的证据,即细胞中是否存在ATP囊泡,体外培养液中能否检测到所释放的ATP。方法:采用出生1～3 d的Sprague-Dawley大鼠,进行体外血管纹缘细胞培养、纯化、鉴定。特异性标记ATP囊泡的喹丫因染色后在荧光显微镜下观察缘细胞中的ATP囊泡。采用生物发光法检测缘细胞细胞外液中所释放的ATP的浓度。结果:体外培养的新生大鼠耳蜗血管纹缘细胞,经流式细胞法检测上皮细胞标志性的角蛋白和波形蛋白的纯度,证实培养所获得的细胞为缘细胞。经喹丫因染色后在荧光显微镜下可见缘细胞细胞质中存在大量的绿色星点状染色。采用生物发光法检测缘细胞细胞外液中ATP的浓度,通过细胞荧光值可计算出ATP的浓度。结论:新生大鼠耳蜗血管纹缘细胞中存在ATP囊泡,并能分泌ATP。     

新生大鼠Klliker器支持细胞体外凋亡及增殖的研究

目的:研究体外Klliker器支持细胞的增殖及凋亡情况,并探讨促进Klliker器支持细胞在体凋亡的可能因素。方法:采用机械分离和酶消化结合的方法提取新生大鼠Klliker器支持细胞并体外培养,通过流式细胞仪检测细胞纯度、凋亡率及凋亡周期,通过MTT法检测该细胞的生长曲线,并运用Realtime PCR及Western blot方法观察不同时间点Caspase-3、Caspase-8、Caspase-9及Bcl-2的表达规律;改变体外培养Klliker器支持细胞培养液中Ca2+、K+及谷氨酸浓度,采用MTT法检测Klliker器支持细胞的存活率。结果:Klliker器支持细胞纯度高达96.56%,呈明显的线性增长;各组细胞的Caspase-3、Caspase-8、Caspase-9及Bcl-2的mRNA及蛋白的表达稳定。提高Klliker器支持细胞外液中Ca2+浓度导致细胞活性降低,而细胞外K+对Klliker器支持细胞活性的影响表现为浓度依赖性,高浓度的谷氨酸对Klliker器支持细胞有一定的保护作用。结论:大鼠Klliker器支持细胞在体外无明显凋亡,呈线性增长趋势。高浓度的Ca2+可能是引起在体Klliker器支持细胞逐渐凋亡的因素,而高浓度的K+及谷氨酸对在体Klliker器支持细胞有一定的保护作用。     

新生大鼠耳蜗K?lliker器在体凋亡的研究

目的：了解不同天龄新生大鼠耳蜗K?lliker器的形态变化,研究凋亡相关因子的mRNA及蛋白的表达水平,探讨K?lliker器在听觉功能发育过程中凋亡的机制。方法选取出生后不同天龄的Sprague－Dawley大鼠共192只,其中出生后1天（P1）、5天（P5）、12天（P12）各6只大鼠耳蜗冰冻切片后,通过苏木精－伊红染色及免疫荧光染色等方法,观察耳蜗K?lliker器的形态结构变化;取 P1大鼠6只行耳蜗基底膜免疫荧光观察;提取出生后1、3、5、7、10、12及14天（P1、P3、P5、P7、P10、P12和P14）大鼠耳蜗基底膜mRNA（各6只）及蛋白（各18只）,运用real－time PCR及蛋白质印迹的方法,观察出生后各天龄组大鼠耳蜗基底膜 K?lliker 器凋亡过程中 bcl－2、caspase－3、caspase－8及caspase－9的表达规律。结果出生后大鼠听力出现之前其耳蜗K?lliker器支持细胞的形态自顶回向底回从高柱状向矮柱状变化,同时支持细胞的数量逐渐减少。出生后不同天龄大鼠耳蜗基底膜的caspase－3、caspase－8、caspase－9及bcl－2的mRNA和蛋白的表达水平均呈明显的时间依赖性。结论在大鼠出生后、听力出现前耳蜗发育的整个阶段,K?lliker器自底回向顶回逐渐发生退化;caspase－3、caspase－8、caspase－9及bcl－2的mRNA和蛋白的表达表现为时间依赖性。     

哺乳动物耳蜗Kölliker器的形态学及退化机制研究

目的　探索哺乳动物耳蜗Kölliker器退化过程中是否存在自噬现象。方法　以P1~P14的SD大鼠耳蜗为研究对象,运用HE染色和透射电镜观察Kölliker器在耳蜗早期发育过 程中的显微和亚显微形态学改变,并利用免疫组化、Western blot分子生物学实验观察自噬标记物LC3、Beclin1、P62在早期发育过程中的动态变化。结果　利用HE染色及透射电镜大鼠耳蜗Kölliker器支持细胞的形态从底回向顶回逐渐由假复层柱状上皮转变为单层立方上皮,细胞层数和数量逐渐减少,最终形成成熟的内沟区域,并观察到自噬小体。免疫组化耳蜗Kölliker器支持细胞LC3-II、Beclin1、P62呈阳性。Western blot观察到P3~P10自噬相关蛋白呈逐渐下降的趋势。结论　哺乳动物耳蜗Kölliker器退化过程中存在时间依赖性的自噬现象。     

三磷酸腺苷在新生大鼠耳蜗血管纹缘细胞中的释放机制

目的探究新生大鼠耳蜗血管纹缘细胞中ATP释放的机制。方法原代培养新生SD大鼠血管纹缘细胞,利用钙离子荧光探针、生物发光法测定ATP浓度和β-氨基己糖苷酶释放率测定法来研究ATP和GPN与P2YR-PLC-IP3通路诱导的内质网钙库反应之间的关系,以及内质网钙库与细胞外ATP浓度和β-氨基己糖苷酶释放率的关系。结果 ATP和GPN可通过P2YR-PLC-IP3通路诱发内质网钙库释放,可被P2YR-PLC-IP3通路拮抗剂抑制。缘细胞外ATP浓度和β-氨基己糖苷酶释放率与细胞内钙离子浓度呈正相关。结论缘细胞外ATP作用于P2Y嘌呤能信号系统触发内质网钙库释放,胞内钙离子诱导溶酶体胞吐作用释放ATP至胞外,从而发挥其生物学作用。     

CICR在哺乳动物耳蜗神经突触传递中的作用

Ca2 作为细胞内的第二信使,几乎参与细胞所有的生理活动,在耳蜗毛细胞的换能、调谐、神经递质释放及基底膜的非线性响应等方面起重要作用.Ca2 在细胞内的浓度受到精细的调控,其中一个重要的调节机制就是Ca2 介导的Ca2 释放(Ca2 -induced Ca2 release,CICR),即细胞外的Ca2 进入胞内,然后诱发了细胞内的钙库释放.     

耳蜗血管纹缘细胞三磷酸腺苷释放机制

三磷酸腺苷（adenosine triphosphate,ATP）被认为是耳蜗中一种重要的信号分子,可以作为共同递质和（或）神经调节物质发挥耳蜗的各种生理功能。目前已证实新生大鼠耳蜗血管纹缘细胞内的ATP囊泡为溶酶体,可以释放ATP。然而,缘细胞释放ATP的机制还未完全阐明,本文结合国内外文献就耳蜗缘细胞中ATP释放机制的研究现状做一综述。     

耳蜗钙通道的研究进展

细胞内钙离子(Ca2 )稳态的维持对生命活动是至关重要的,细胞变性坏死可能与胞内Ca2 超载有关。细胞内Ca2 水平的调节通过以下途径实现:钙通道、Na -Ca2 交换、钙泵以及细胞内部的Ca2 摄取与释放(线粒体和肌浆网等)。近年来许多研究已表明,耳蜗毛细胞和螺旋神经节细胞等细胞膜上     

Neurogenesis in the superior olivary complex in the rat

In a previous paper we provided evidence that crossed projection neurons are generated earlier than uncrossed projection neurons in the lateral superior olive. The aim of the present study was to determine if other major nuclei of the superior olivary complex (SOC), the medial superior olivary (MSO), the superior paraolivary (SPN) and the medial trapezoid (MTB) nuclei, are distinguished by their neuronal constitutions of birthdates. Pregnant rats were injected intraperitoneally with 5-bromodeoxyuridine (BrdU), the thymidine analogue, to label the neurons on one of the embryonic (E) days E11-E16. When the progeny rats reached adulthood, the brains were processed for BrdU immunohistochemistry. The MSO was mostly composed of neurons generated on E12 (95%). The remaining neurons in the MSO completed their neurogenesis by E13. The SPN neurons were generated from E12 to E14 with a peak on E13 (80%). Regardless of the morphological heterogeneity, the SPN consisted of a single population of neurons in terms of neurogenesis. The MTB neurons were generated from E13 to E16 with a peak on E14 (73%). In contrast to the previous assumption, no topographical relationship existed between neurogenesis and tonotopicity within each nucleus of the SOC.     

Cation transport in the ampulla of the semicircular canal and in the endolymphatic sac

Summary We examined the effects of anoxia and ethacrynic acid on the endolymphatic potential and cation activity in the superior ampulla of the guinea pig, using double-barrelled ion-exchanger microelectrodes. In normal guinea pigs the ampullar endolymphatic potential was +3.9±1.2 mV (n=32), the Cl^– activity 130±4.6 mM (n=9), and the Na⁺ activity 18.4±4.4 mM (n=20). After anoxia, the ampullar DC potential decreased rapidly and reversed its polarity within 5 min. It then decreased gradually for 60 min and increased afterwards to approximately zero. K⁺ activity decreased gradually after a latency of 10 min, whereas Na⁺ activity increased. During the gradual decrease of a negative ampullar endolymphatic potential, an increase in Na⁺ activity was observed. Thirty minutes after the intravenous injection of ethacrynic acid (100 mg/kg), the potential began to decrease, changed to a negative polarity, and approached a maximum negative level 100 min after the injection. The decrease in K⁺ activity corresponded to the reduction of potential whereas Na⁺ activity remained unchanged. The DC potential of the endolymphatic sac in normal guinea pigs was + 14.7±5.1 mV (n=17). The Na⁺ concentration was 103.3±14.7 mM (n=14) and the K⁺ concentration was 11.6 ±0.8 mM (n=4). After anoxia, the DC potential decreased rapidly and approached 0 mV within 8 min. No negative potential could be observed. The Na⁺ concentration began to increase 2 min after anoxia and reached the extracellular Na⁺ concentration about 30 min later. No significant effect of intravenous administration of ethacrynic acid (100 mg/kg) on DC potential and Na + concentration could be observed. The results suggest the presence of a different ion transport system in the endolymphatic sac from that of the cochlea and the ampullae of the semicircular canals.     

Chasing the dragon in the ear

In the UK, the prevalence of drug misuse has risen in the last decade. Otorhinolaryngological surgeons need to be aware that small amounts of illegal substances, such as heroin, cannabis and cocaine, may be hidden in the external auditory meatus. We report, for the first time, concealment of heroin in the external auditory meatus. The medico-legal and ethical considerations are also discussed.     

Temporal synchronization in the primary auditory response in the pigeon

Spike potentials were recorded from single fibres in the auditory nerve of the pigeon. In responses elicited by tonal stimuli, the timing of each spike relative to stimulus waveform was measured and period histograms were constructed. Phase locking of spikes was estimated in terms of a synchronicity index obtained by vector addition within the period histogram. A second measure of synchrony in the spike responses was obtained, that of temporal dispersion. For a population of fibres, vector strength of phase locking decreased for frequencies above 1 kHz, as reported for several other species. Temporal dispersion, however, also decreased with frequency, indicating enhanced temporal synchrony as frequency increased within the bandwidth of phase locking. The upper frequency limit of phase locking appears to depend on irreducible jitter of biological origin in the timing of spikes. For individual fibres, the bandwidth of synchronization of spikes consistently exceeds the response area, covering in addition the areas of suppression adjacent to the response area. Spike trains suppressed by a tonal stimulus become synchronized to that stimulus. Phase angles of synchronized responses systematically change as a function of tone level, when tone frequency is above or below CF, as reported for other avian species. Synchronicity and phase angle intensity functions are quite independent of spike rate intensity functions.