【作者】 于宁；

【导师】 翟所强； 赵红波； 杨仕明；

【作者基本信息】 中国人民解放军军医进修学院 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 随着聋病机制研究的快速发展,对耳蜗主动放大机制的探索成为理解耳聋病发生机制的最关键的一环。以Prestin(Motor Protein,动力蛋白)为分子基础的外毛细胞电运动(Electromotility)则是耳蜗主动放大机制形成的必要条件。1985年Brownell发现了细胞长度能随电位的变化快速改变的外毛细胞电运动。外毛细胞这种独一无二的外电运动的频率高达110kHz,且不直接依赖于ATP与钙离子的浓度,能够对声波引起的耳蜗基底膜的振动进行放大和修饰,从而大大提高了内耳敏感性和频率选择性。细胞膜低通滤波特性与高频电运动之间的差异提示,外毛细胞压电反应(Piezoelectricity)在耳蜗机电转换过程可能具有重要作用。2000年发现了编码膜动力蛋白(Prestin)的基因Pres,随后的工作证实了Prestin蛋白是外毛细胞电运动及压电反应所必须的分子基础。自Prestin蛋白发现以来,其在听觉调控机制中的作用及基本特性一直就是听觉领域中的研究热点。受各种因素的制约,目前尚缺乏Prestin的在外毛细胞侧膜中的存在形式、位置及更新机制的直接实验证据。虽有药物调控外毛细胞电运动的报道,但未见Prestin的表达能否被调控的报道。为了探索Prestin及对听觉的调控机理,本研究试以Prestin蛋白在外毛细胞膜上的表达更新为切入点,综合利用病理生理等技术方法,对外毛细胞电运动的调控机制及压电反应的意义进行初步研究,并结合实验结果从提高听觉灵敏度与频率选择性的角度对Prestin蛋白及其表达调控机制进行分析讨论。第一部分耳蜗外毛细胞基底细胞膜Prestin蛋白分布与表达的研究目的:本研究以明确OHC基底细胞膜上是否存在Prestin的表达及其在侧膜三层结构中的定位为目标,对Prestin蛋白的功能及可能的更新机制进行了探索。Prestin是外毛细胞电运动的分子基础,特异性地表达在OHC侧膜上,在胞浆、表皮板及纤毛上无表达。以往工作虽在外毛细胞膜中发现有高密度的蛋白颗粒分布,但未能确定这些蛋白颗粒与Presin蛋白之间的关系及在基底细胞膜上的有无分布。方法:在本研究中,分别取正常小鼠、大鼠及豚鼠耳蜗的单离外毛细胞及全基底膜,在激光共聚焦显微镜下,利用Prestin特异性抗体与细胞膜标记di-8-ANEPPS双染,对Prestin在两种标本上的表达与分布情况进行了观察。结果:(1)不仅OHC基底侧膜上存在Prestin抗体的阳性染色,OHC底端细胞膜上亦见阳性染色,但其荧光强度仅为侧膜的80.5%和61.1%;(2)与细胞膜标记di-8-ANEPPS染色相比,Prestin抗体的阳性染色位于细胞膜的外侧;经低渗细胞外液处理后,OHC侧膜中的质膜层可与SSL和CL分离,在激光共聚焦显微镜下,外毛细胞膜最外侧的质膜层上可见明显的Prestin抗体阳性染色。结论:通过对本研究结果的分析,我们认为Prestin分布于外毛细胞膜中最外侧的质膜层上,在细胞基底部细胞膜上亦有表达。第二部分Prestin蛋白表达的功能性调控目的:急性使用水杨酸钠能够可逆性的抑制外毛细胞的电运动并引起听力下降,但长期服用水杨酸钠可使畸变产物耳声发射幅值增大。因Prestin是外毛细胞电运动的动力蛋白,明确长期服用水杨酸钠对Prestin表达及对外毛细胞电运动有无直接影响,并提示耳蜗放大机制有着重要意义。方法:本部分中将按照经典的耳鸣建模方法,使用水杨酸钠建立动物模型。在记录耳声发射及外毛细胞电运动的基础上,结合实时定量PCR及Western blot等方法,在RNA、蛋白质、细胞以及整体动物等多个水平上,探索Prestin表达与耳蜗功能之间的关系。结果:长期服用水杨酸钠不仅增大了畸变产物耳声发射幅值,也明显提高了Prestin的表达与外毛细胞电运动能力。在mRNA与蛋白水平,长期服用水杨酸钠使Prestin蛋白表达量增大至正常水平的3～4倍。外毛细胞电运动直接相关的充电密度提高了18%。此类变化是可逆性,停止服用水杨酸钠后Prestin的表达与外毛细胞电运动则恢复至正常值。本研究从病理生理的角度证明了Prestin对听觉灵敏度及频率选择性的调控作用。值得注意的是,在这一过程中虽然环氧合酶(Cox)Ⅱ的表达不受影响,但是NF-KB的表达被抑制,而核转录因子c-fos和egr-1的表达则被上调。结论:在体状态下长期注射水杨酸钠后随着耳蜗功能的增强,Prestin蛋白表达动态上调。Cox-Ⅱ依赖性通路对调控Prestin表达有重要作用。第三部分Prestin蛋白压电效应提高外毛细胞的高频响应目的:听觉换能过程中,哺乳动物活体耳蜗外毛细胞在产生感觉器电位的同时亦承受因基底膜振动所带来的机械刺激。在外力刺激下,耳蜗外毛细胞基于Prestin产生电运动的同时还具有压电反应,并通过调控基底膜的行波来有效提高听觉敏感度与频率选择性,这是耳蜗主动放大机制最重要的生理基础。电压依赖性的外毛细胞电运动可被跨膜电位驱动。但通常细胞膜因膜电容的存在而成为一个截止频率为1kHz左右的低通滤波器,尽管电运动的分子基础—Prestin蛋白构型改变的频率高达100kHz,但其驱动力—跨膜电位的高频部分可被低通滤波器大幅度衰减。根据机械性牵拉或压缩外毛细胞能够引起细胞膜电位变化并有相应压电电流产生的现象,虽推测压电反应可能是其克服高频衰减的能量来源,但尚缺乏直接地实验证据。方法:将利用全细胞膜片钳记录外毛细胞压电反应的方法,在不同条件下给予外毛细胞不同频率的机械刺激来记录外毛细胞的压电反应,并通过与模拟电路以及支持细胞高频响应的比较,来验证Prestin蛋白压电效应是否能够提高外毛细胞的高频响应。本部分工作将利用全细胞膜片钳记录外毛细胞压电反应的方法,在不同条件下给予外毛细胞不同频率的机械刺激来记录外毛细胞的压电反应,并通过与模拟电路以及支持细胞高频响应的比较,来验证Prestin蛋白压电效应是否能够提高外毛细胞的高频响应。结果:本研究发现外毛细胞压电效应的存在使得外毛细胞的高频频响显著增大,并将截止频率扩展至70-90kHz。实验中采用全细胞膜片钳可在外毛细胞上记录到高达80kHz的频响平台。当去除压电效应后外毛细胞对单纯电刺激的反应呈低通特性,截止频率仅为1kHz左右,这与其它组织细胞相类似。结论:本研究结果证实在耳蜗主动放大机制中外毛细胞电运动可增大高频响应并有效扩展哺乳动物的听觉频率范围。第四部分耳蜗Deiters细胞及缝隙连接对外毛细胞电运动的调控目的:哺乳动物的听觉器官对声音具有高灵敏和极精细的分辨能力,以外毛细胞电运动为特征的耳蜗主动放大机制是其重要的基础。耳蜗外毛细胞处于支持细胞,特别是Deiters细胞(Deiters cell,DC)的支撑和环绕之中。Deiters细胞细长的指突与外毛细胞顶部的表皮板相连,宽大的胞体与外毛细胞底部相连接构成稳定的弓形结构以支撑Cortis器的外部形态。Deiters细胞与外毛细胞之间虽有组织连接,但以往并未观察到两者之间存在直接电导联的证据。方法:利用双膜片钳技术在单离的外毛细胞—Deiters细胞对上,对Deiters细胞及细胞间的缝隙连接通道耦合对外毛细胞电运动影响进行观察。结果:当给予Deiters细胞适当的电压或电流刺激时能够直接影响外毛细胞电运动状态。Deiters细胞去极化时能够降低与外毛细胞电运动直接相关的NLC及频率响应。当Deiters细胞与外毛细胞间的机械耦合或Deiters细胞的细胞骨架被破坏后,Deiters细胞对非线性电容的影响消失。同时我们观察到Deiters细胞间的缝隙连接(Gap junction,GJ)被破坏后可使膜电位及NLC的电压依赖特性发生改变。另外与外毛细胞连接在一起的Deiters细胞可以提高ATP对外毛细胞电运动调控的效能。结论:Deiters细胞能够通过细胞骨架或改变细胞膜的张力来直接影响外毛细胞的电运动。Deiters细胞间的缝隙连接对外毛细胞状态有着直接的调控作用。上述研究结果将为我们更深入地理解听器精细调控及缝隙连接在内耳中的功能提供新的思路。通过上述研究,我们证实在外毛细胞基底部细胞膜上有Prestin的表达,并且Prestin更新的起始位点可能就在这一区域。基于Prestin的压电反应则相应提高了听觉器官对高频频率的选择性及灵敏度。Prestin的表达与Deiters细胞状态能够有效的调控外毛细胞电运动,从而提高听觉灵敏度。而且综合分析来自RNA、蛋白质、细胞及整体水平的数据后,可以证明Prestin的表达受耳蜗功能的调控并具有功能依赖性的特点。更多还原

【Abstract】 Siting above the basllar membrane,outer hair cells(OHCs) appear able to perceive its vibration through their mechanosensitive hair bundles and to feed back mechanical forces that enhance both auditory system’s sensitivity and its frsquency selectivity.They are mechanical components of the cochlea and have been the subject of many experiments designed to discover just exactly how they generate mechanical forces and how these forces contribute to the micromechanics of the cochlea.The electromotilty of the cell body has been the subject of the most extensive studies,which is a high-frequency length change of up to 5%that can be driven at acoustic frequencies and that it is not directly dependent on ATP or calcium ion but it does require the cytoplasm to have positive turgor pressure.This electromotilty is both fast enough and strong enough to sharpen the sound-induced mechanical displacements of the basilar membrane.OHC electromotility is a direct transduction of cell length change to membrane potential and is functionally equivalent to the transduction piezoelectric.Prestin is required for OHC motility and plays a central role in OHC electromotility.Today,we still have no knowledge of Prestin’s most nature,such as the three dimensional organization.Without definitive information,it is difficult to understand how is alters electromechanical coupleing the OHC PM, how is involved in the mammalian cochlear amplifier. Part one:Prestin is expressed on the whole outer hair cell basolateral surfacePrestin has been identified as a motor protein responsible for outer hair cell (OHC) electromotility.Previous experiments revealed that OHC electromotility and its associated nonlinear capacitance resided in the OHC lateral wall and was not detected at the apical cuticular plate and basal region.In this experiment,the distribution of prestin in adult mouse,rat,and guinea pig OHCs was re-examined by use of immunofluorescent staining and confocal microscopy.We found that prestin labeling was located at the whole OHC basolateral wall,including the basal plasma membrane.However,staining at the basal membrane was weak.As compared with the intensity at the lateral wall,the intensities of prestin labeling at the membrane at the nuclear level and basal pole were 80.5%and 61.1%, respectively.Prestin labeling was not found at the cuticular plate and stereocilia. The prestin labeling was also absent in the cytoplasmand nuclei.The OHC lateral wall above the nuclear level is composed of the plasmamembrane,cortical lattice, and subsurface cisternae.By costaining with di-8-ANEPPS,prestin labeling was found at the outer layer of the OHC lateral wall,which was further evidenced by use of a hypotonic challenge to separate the plasma membrane from the underlying subsurface cisternae.The data revealed that prestin is expressed at the whole OHC basolateral membrane.Prestin in the basal plasma membrane may provide a reservoir on the OHC surface for prestin-recycling and may also facilitate performing its hypothesized transporter functionPart two:Prestiu up-regulation in chronic salicylate(aspirin) administration: An implication of functional dependence of prestin expressionSalicylate(aspirin) can reversibly eliminate outer hair cell(OHC) electromotility to induce hearing loss.Prestin is the OHC electromotility motor protein.Here we report that,consistence with increase in distortion product otoacoustic emission,long-term administration of salicylate can increase prestin expression and OHC electromotility.The prestin expression at the mRNA and protein levels was increased by three- to fourfold.In contrast to the acute inhibition,the OHC electromotility associated charge density was also increased by 18%.This incremental increase was reversible.After cessation of salicylate administration,the prestin expression returned to normal.We also found that long-term administration of salicylate did not alter cyclooxygenase(Cox)Ⅱexpression but down-regulated NF-kB and increased nuclear transcription factors c-fos and egr-1.The data suggest that prestin expression in vivo is dynamically up-regulated to increase OHC electromotility in long-term administration of salicylate via the Cox-Ⅱ-independent pathways.Part three:Piezoelectricity Increases Outer Hair Cell High Frequency ResponseOuter hair cell(OHC) electromotility is a cochlear amplifier and can actively boost the basilar membrane vibration to enhance auditory sensitivity and frequency selectivity.OHC electromotility is membrane-potential dependent and driven by cross-membrane voltage.Although the conformation of prestin motor proteins can be rapidly changed up to 100 kHz,its driving force(cross-membrane voltage) would be dramatically attenuated at high frequency by membrane capacitance,which forms a lowpass filter with cut-frequency less than 1 kHz. Outer hair cells also have remarkable piezoelectricity.Mechanically elongating and compressing OHC can produce electric currents.Here,we report that OHC piezoelectricity can overcome membrane capacitance damping to improve OHC high frequency responses.The OHC piezoelectric response showed a high-pass property and was increased as the stimulus frequency was increased.The cutfrequency was 70-90 kHz,mainly limited by the recording system. Simultaneous administrations of electronic and mechanical(piezoelectric) stimulation to the OHC,which mimics the OHC suffered electronic(receptor current through transduction channels) and mechanical(the vibration of the basilar membrane) stimulations in vivo,generated the flat response up to 80 kHz. Abolishment of piezoelectricity eliminated this high frequency enhancement.Like a regular cell,the sole electronic frequency response of the OHC was low-pass; the cutfrequency was～1 kHz.Finally,as computer modeling expected,the resonant peaks were also visible in the responses to electronic-mechanical stimulation.Our results indicate that OHC electromotility can perform at high frequency effectively to contribute active cochlear mechanics in whole mammalian auditory frequency range.Part four:Contribution of cochlear supporting cells and gap junctional coupling to control of hearing sensitivity in the inner earDelicious mammalian hearing function relies upon outer hair cell(OHC) electromotility to increase hearing sensitivity and frequency selectivity.In situ, OHCs are constrained by Deiters supporting cells.Here,we report that in addition to support function Deiters cells(DCs) can induce OHC movement and mediate OHC electromotility.DCs had no direct electrical conductance with OHCs. However,voltage and current stimulations in DCs could affect OHC electromotility.Depolarization of DCs reduced OHC electromotility associated nonlinear capacitance(NLC) and frequency responses.Breaking DC-OHC mechanical coupling or destroying DC cytoskeleton abolished this effect.We also found that uncoupling of gap junctional coupling between DCs could induce large changes in the membrane potential and current and shifted the NLC voltage dependence.DCs could also enhance the ATP effect on OHC electromotility. These data suggest that DCs can directly mediate OHC electromotility through the cytoskeleton to alter membrane tension.These findings also provide new understandings of hearing control and gap junctional function in the inner ear.The above results imply that basal plasma membrane of OHC may provide a reservoir on the OHC surface for prestin,Prestin’s expression can be modulated. These data also suggest that DCs can directly mediate OHC electromotility through the cytoskeleton to alter membrane tension and piezoelectricity of OHC enhaces voltage changes at high frequencies that may contribute to auditory system’s sensitivity and its frsquency selectivity.更多还原