利诺吡啶对豚鼠耳蜗外毛细胞和支持细胞钾电流的影响

目的 观察KCNQ家族钾通道特异性阻滞剂利诺吡啶(linopirdine)对豚鼠耳蜗单离外毛细胞和Deiters细胞(支持细胞)总钾电流的影响，初步探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的分布。方法 运用膜片钳技术，在全细胞模式下记录正常细胞外液中8个外毛细胞和5个Deiters细胞的总钾电流，并观察100μmol／L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果 在正常细胞外液中，单离外毛细胞可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流(the K^ current activated at negative potential，IKn)两种钾电流，而单离Deiters细胞中只记录到外向整流性钾电流。加入100μmol／L利诺吡啶后，外毛细胞中的四乙基二乙胺敏感的钾电流减小，IKn被完全抑制；而Deiters细胞中的外向整流性钾电流大小无变化。结论 KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞，其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分，并构成全部的IKn；但KCNQ家族钾通道不存在于豚鼠耳蜗Deiters细胞。     

川芎嗪对庆大霉素耳中毒耳蜗外毛细胞和血管纹的保护作用

目的：探讨在庆大霉素（gentamycin，GM）耳中毒情况下，川芎嗪（tetramethylpyrazine，TMP）对豚鼠耳蜗外毛细胞和血管纹边缘细胞的保护作用。方法：12只豚鼠随机分为GM组、联合用药组、TMP组及对照组，用药十天后处死，采用透射电镜观察耳蜗外毛细胞及血管纹边缘细胞超微结构，扫描电镜观察血管纹边缘细胞表面形态。结果：透射和扫描电镜显示，联合用药组外毛细胞及血管纹边缘细胞超微及表面结构破坏不均明显轻于庆大霉素组，特别是其中的线粒体结构破坏与数目减少更显著轻于庆大霉素组。结论：川芎嗪具有保护庆大霉素耳中毒耳蜗外毛细胞和血管纹结构的作用，从而拮抗庆大霉素耳毒性。     

小鼠耳蜗螺旋神经节细胞电生理学特性的研究

目的：应用膜片钳技术记录小鼠耳蜗螺旋神经节细胞的全细胞电流,了解电压依赖性离子通道的基本电生理学特性,并比较耳蜗顶、底转螺旋神经节细胞电生理学特性的差异。方法：应用全细胞构型电压钳制技术,采用不同的电极内液及阻断剂,在不同的刺激参数下记录耳蜗顶、底转螺旋神经节细胞的电压依赖性离子通道电流,并进行分析比较。结果：实验记录到了内向的钠电流、延迟整流钾电流、超极化激活内向阳离子通道电流及瞬时外向钾电流,并发现耳蜗顶、底转螺旋神经节细胞的延迟整流钾电流及瞬时外向钾电流的电生理学特性具有显著性差异（P〈0．05）。结论：实验记录到的各种离子电流数据表明耳蜗螺旋神经节细胞具有完成动作电位的形成、传导并对其功能进行调节的离子通道基础;耳蜗顶、底转螺旋神经节细胞电生理学特性的差异有助于听觉的形成过程。     

组织块培养法及胶原酶消化法分离培养豚鼠耳蜗微血管内皮细胞

目的：建立稳定的豚鼠耳蜗微血管内皮细胞的培养方法。方法：采用显微解剖技术分离出豚鼠耳蜗血管纹,分别用组织块法及酶消化法进行培养及纯化。结果：耳蜗血管纹组织块培养后2天,部分组织块边缘有散在的细胞生长,这些细胞增逐渐增殖形成大片细胞集落;耳蜗血管纹组织经胶原酶消化后1－3天,可观察到培养皿底由一些细胞组成的细胞岛;细胞纯化后大多呈多边形,致密融合时具有内皮细胞培养时典型的“铺路石样”外观,经免疫组化检测其内皮细胞标志性抗原Ⅷ因子,95％以上的培养细胞的胞浆中呈棕黄色阳性反应。结论：组织块法及酶消化法能够获得体外培养的豚鼠耳蜗微血管内皮细胞。     

顺铂对小鼠耳蜗螺旋神经节细胞延迟整流钾电流的影响

目的探讨耳毒性药物顺铂对耳蜗螺旋神经节细胞延迟整流钾电流的影响,了解顺铂耳毒性的离子机制。方法选择状态良好培养2—14d的耳蜗螺旋神经节细胞在电压钳制条件下记录的钾电流作为对照组,浴液中分别加入100,500,1000,5000μM不同浓度的顺铂溶液后记录的电流为实验组,观察其对钾通道电流电生理学特性的影响,并观察冲洗后电流的恢复情况。结果实验显示耳蜗螺旋神经节细胞钾通道电流的电生理学特性受不同浓度顺铂溶液的影响,并具有浓度依赖性,表现在其活化动力学和电流幅度的改变。本实验中顺铂的50％的电流被抑制时的浓度（IC50）为294μM,冲洗后钾通道电流逐渐恢复。结论顺铂可以改变耳蜗螺旋神经节细胞钾通道电流的电生理学特性,这种变化在短期内可逆,揭示其耳毒性的形成具有一定的离子机制。     

TMEM16A对衰老耳蜗血管纹周细胞凋亡的作用

目的 原代培养豚鼠耳蜗血管纹毛细血管周细胞(PCs),探讨TMEM16A在耳蜗血管纹PCs上的表达变化及其对衰老耳蜗血管纹PCs凋亡的影响.方法 原代培养耳蜗血管纹PCs并鉴定;运用β-半乳糖苷酶染色确定细胞衰老模型;全细胞膜片钳技术记录PCs上CaCCs的电流变化;免疫荧光检测耳蜗血管纹PCs上TMEM16A的表达变...     

利诺吡啶对豚鼠耳蜗外毛细胞和支持细胞钾电流的影响

目的　观察KCNQ家族钾通道特异性阻滞剂利诺吡啶 (linopirdine)对豚鼠耳蜗单离外毛细胞和Deiters细胞 (支持细胞 )总钾电流的影响 ,初步探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的分布。方法　运用膜片钳技术 ,在全细胞模式下记录正常细胞外液中 8个外毛细胞和5个Deiters细胞的总钾电流 ,并观察 10 0 μmol/L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果　在正常细胞外液中 ,单离外毛细胞可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流 (theK+ currentactivatedatnegativepotential,IKn)两种钾电流 ,而单离Deiters细胞中只记录到外向整流性钾电流。加入 10 0 μmol/L利诺吡啶后 ,外毛细胞中的四乙基二乙胺敏感的钾电流减小 ,IKn被完全抑制 ;而Deiters细胞中的外向整流性钾电流大小无变化。结论KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞 ,其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分 ,并构成全部的IKn;但KCNQ家族钾通道不存在于豚鼠耳蜗Deiters细胞。     

噪声对耳蜗边缘细胞及螺旋神经节细胞乙酰化组蛋白H2B的影响

目的　观察噪声性聋豚鼠耳蜗侧壁血管纹边缘细胞及蜗轴螺旋神经节细胞乙酰化组蛋白H2B表达水平的变化。方法　成年雄性白色红目豚鼠50只随机分为噪声组和对照组,通过听性脑干反应检测两组豚鼠噪声前听力,噪声组给予高强度窄带噪声（122 dB SPL,3 h）暴露,于暴露后1 h行ABR检测噪声后听力,并通过免疫荧 光染色和Western blot方法检测暴露后2 h两组豚鼠耳蜗血管纹边缘细胞及蜗轴螺旋神经节细胞中乙酰化组蛋白H2B的表达水平。结果　两组豚鼠噪声暴露前听力在4、8、16与32 kHz频率的反应阈比较,差异无统计学意义。噪声组在噪声刺激1 h后ABR阈值在4、8、16与32 kHz频率的最大输出90 dB无反应。免疫荧光染色显示在噪声刺激2 h后血管纹边缘细胞中乙酰化组蛋白H2B荧光强度明显下降,Western blot显示噪声组豚鼠侧壁中乙酰化组蛋白H2B表达（H2B-AcK5/β-actin的比值为0.3471±0.0843）较对照组（0.6114±0.0207）明显下调,两组比较差异有统计学意义（t =5.268,P＜0.01）。耳蜗蜗轴组织中乙酰化组蛋白H2B表达在噪声组和对照组中分别为（0.4993±0.0994）和（0.5139±0.0132）,两组比较差异无统计学意义（P ＞0.05）。结论　噪声可导致豚鼠耳蜗血管纹边缘细胞组蛋白乙酰化水平显著下降,螺旋神经节细胞中组蛋白乙酰化水平无明显改变,血管纹边缘细胞组蛋白乙酰化失衡有可能参与噪声性聋的发生发展。     

豚鼠耳蜗单离Deiters细胞的钾电流

目的 研究豚鼠耳蜗单离Ｄｅｉｔｅｒｓ细胞的钾电流及其特性。方法 运用膜片钳技术,在全细胞模式下记录正常细胞外液中钾电流,不同Ｋ＾＋浓度的细胞外液对细胞反转电位和外向钾电流的影响,四氨基吡啶（４－ａｍｉｎｏｐｙｒｉｄｉｎｅ,４－ＡＰ）和四乙工胺（ｔｅｔｒａｅｔｈｙｌａｍｍｏｎｉｕｍ,ＴＥＡ）对钾电流成分的阻滞作用,探讨外向钾流通道的激活和失活动力学。结果 单离Ｄｅｉｔｅｒｓ细胞具有电压依赖的外向整流     

杂色和白色豚鼠听反应阈及耳蜗血管纹细胞增殖活？…

目的 研究染色和白色成年豚鼠正常听反应阈及耳蜗血管纹细胞增殖活性的差异。方法 用听觉诱发电位仪分别测定杂色和白色豚鼠４０Ｈｚ听觉相关电位（４０Ｈｚ ＡＦＲＰ）及听性脑干反应（ＡＢＲ）阈值，用免疫组化法检测两种豚鼠耳蜗血管纹增殖细胞核抗原（ＰＣＮＡ）表达的差异。结果 杂色和白色豚鼠耳蜗血管纹中间细胞ＰＣＮＡ染色均呈阳性，但前者ＰＣＮＡ的表达显著强于后者；两种豚鼠的听反应阈值无显著性差异。结论 正常染     

Mechanisms of endocochlear potential generation by stria vascularis

It is commonly accepted that the endocochlear potential (EP) of the cochlea is generated by an electrogenic transport of potassium into scala media by the marginal cells of stria vascularis. We have studied the potential and potassium concentration gradients as stria vascularis was penetrated with double-barreled potassium selective electrodes in the guinea pig cochlea. Our data demonstrate that a region exists in stria which is positively polarized (higher than the EP), but which has a low (perilymph-like) potassium composition. It is concluded that EP cannot be generated by the marginal cells alone but may involve passive potassium movement across the apical membranes of the basal cells. A model is presented which is consistent with many anatomical and physiological features of stria vascularis.     

噪声刺激对耳蜗一氧化氮合酶的影响

目的：探讨一氧化氮（ＮＯ）在噪声性聋发病中的作用。方法：用中高频连续稳态噪声制作噪声性聋的动物模型,用ＮＡＤＰＨ－黄递酶组织化学、原位杂效和Ｎｏｒｔｈｅｒｎ印迹法,观察噪声刺激对耳蜗一氧化氮合酶（ＮＯＳ）表达的影响。结果：组织化学法显示ＮＯＳ主要分布于内外毛细胞、螺旋神经节细胞和血管纹边缘细胞;原位杂效法发现ＮＯＳｍＲＮＡ在内外毛细胞、螺旋神经节细胞胞浆内均可见阳性染色,但血管纹边缘细胞无阳性染色     

Ultrastructure of the stria vascularis and Reissner's membrane in experimental hydrops

A time-sequence study was made of the early ultrastructural changes of the stria vascularis and Reissner's membrane in the guinea pig after obliteration of the endolymphatic sac and duct. Pathological alterations of both the stria vascularis and Reissner's membrane were found to start in the apex of the cochlea. The morphological changes of the stria vascularis were characterized by an increase of vesicles in the marginal cells and by intercellular edema, followed by vacuolization and atrophy of marginal and intermediate cells. In Reissner's membrane extensive gaps in the mesothelial cell layer were observed together with intracellular pathology of the epithelial cells. The significance of these ultrastructural changes in the stria vascularis and Reissner's membrane with regard to the pathophysiology of the endolymphatic hydrops is discussed.     

耳蜗血管纹组织块的缘细胞培养

目的 :建立豚鼠耳蜗血管纹 (SV)组织块缘细胞 (MCs)的培养方法 ,为进一步研究药物耳毒性及其作用机制奠定基础。方法 :2 6只豚鼠按SV培养时间随机分成 4组 :2 4h组 (n =8) ;72h组 (n =8) ;>72h组 (n =8) ;对照组 (新鲜SV固定组 ,n =2 )。显微解剖数段连同螺旋韧带的SV组织块 ,置于 5 %CO2 / 95 %空气的二氧化碳恒温 (37℃ )培养箱中进行培养 ,分别进行形态学和组织学观察。结果 :培养 2 4hSV组织块保持良好活性 ,其组织学结构与新鲜固定的SV结构无明显差异 ;培养 72hSV组织块与新鲜固定的SV在组织学结构方面有显著性差异 ,不能观察到正常的SV结构 ,组织结构松散 ,缘细胞从组织块离心性生长出来 ;从SV组织块培养出的缘细胞能在培养皿内存活 13d。结论 :采用组织块培养技术 ,成功地建立了豚鼠耳蜗SV组织块的缘细胞培养方法 ;培养 2 4h的SV组织块光镜下保持了良好活性和正常组织学结构 ,可用来进一步研究药物耳毒性及其作用机制。     

Ototoxic effect of potassium canrenoate on the guinea pig cochlea

Potassium canrenoate (PC) is a diuretic with antialdosterone action, reducing the reabsorption of sodium in the efferent renal tubules. This drug has been reported to damage only the marginal cells of the stria vascularis in the cochlea. The perilymphatic space of the guinea pig cochlea was perfused with an artificial perilymph containing 5 x 10(-3) M potassium canrenoate. Following the onset of perfusion, the endocochlear dc potential (EP) gradually declined to around 10 mV but did not become negative even when the perfusion was continued. A similar decrease in EP was observed in kanamycin-deafened guinea pigs. When the respirator was turned off and perfusion discontinued, a large negative EP appeared during 5 min of anoxia in normal guinea pigs but not in the kanamycin-deafened guinea pigs. The decreased EP recovered to preanoxic level after resumption of ventilation. The cochlear microphonics (CM) also gradually declined in parallel with the EP. The summating potential (SP) showed only a minor change. The potassium ion activity in the endolymph decreased slightly but the sodium ion activity remained unchanged during perfusion. These findings suggest that the main target of the PC is the cells of the stria vascularis.     

谷氨酸-天冬氨酸转运体在豚鼠耳蜗的分布

目的研究谷氨酸-天冬氨酸转运体(glutamate—aspartate transporters，GLAST)在正常豚鼠耳蜗内的分布，为探讨GLAST在防止耳蜗谷氨酸(Glu)神经毒性中的作用提供形态学基础。方法选取健康红目豚鼠6只，采用免疫组织化学方法，以山羊抗GLAST抗体为标记物，观察正常豚鼠耳蜗中GLAST的表达及分布。结果在正常豚鼠耳蜗的内、外毛细胞，内、外毛细胞周围的支持细胞，螺旋神经节细胞，血管纹边缘细胞和螺旋缘上皮，均有GLAST阳性表达。结论GLAST在正常豚鼠耳蜗内主要分布于内、外毛细胞，内、外毛细胞周围的支持细胞，螺旋神经节细胞、血管纹边缘细胞和螺旋缘上皮，其功能尚需进一步的研究。     

Marginal cells of the stria vascularis in vitro]

Explants of stria vascularis and spiral ligament of the guinea pig cochlea were cultivated and after 2 days fibroblast-like cells were found growing around the explant. Marginal cells advanced at 15 microns/day to the border of the explant, and after 2 weeks they proliferated on top of a thin layer of fibroblast-like cells outside the explant, replacing several layers of fibroblast-like cells. Tight junctions and interdigitations of the lateral membranes were found between all neighbouring marginal cells. Their apical surface was covered by microvillus-like membrane extensions. The basal membrane of the new marginal cells did not interdigitate with the underlying membranes of fibroblast-like cells; there was always a gap between the two cell types. The results demonstrate that marginal cells of the stria vascularis are capable of repairing damage to the epithelium, such as may be caused by endolymphatic hydrops, even if the luminal side contains perilymph-like fluid. Furthermore, the cell culture allows living, clearly identified marginal cells to be studied in vivo.     

Ultrastructural localization of Ca2+-binding sites in the spiral limbus,the stria vascularis and Reissner's membrane of the guinea pig

Summary Cations were precipitated with potassium antimonate in the cochlea of the guinea pig and electron microscopy was used to analyze the distribution of the formed reaction products. Differences in precipitate density between neighboring cells in Reissner's membrane, in the stria vascularis and in the limbus are described. Electron spectroscopic imaging was performed to obtain information about the spatial distribution of the precipitates and their elemental composition.     

Ultrastructural localization of Ca2+-binding sites in the spiral limbus, the stria vascularis and Reissner's membrane of the guinea pig

Cations were precipitated with potassium antimonate in the cochlea of the guinea pig and electron microscopy was used to analyze the distribution of the formed reaction products. Differences in precipitate density between neighboring cells in Reissner's membrane, in the stria vascularis and in the limbus are described. Electron spectroscopic imaging was performed to obtain information about the spatial distribution of the precipitates and their elemental composition.     

噪声对豚鼠耳蜗血管纹紧密连接蛋白claudin-5表达及血迷路屏障通透性的影响

目的研究噪声对豚鼠耳蜗血管纹紧密连接蛋白claudin-5表达及血迷路屏障功能的影响。方法40只豚鼠随机分为正常对照组(20只)和噪声暴露组(20只),噪声暴露组给予声强为115dB SPL白噪声暴露,每天6小时,连续两天,对照组不予噪声暴露。于实验前及噪声暴露后次日对两组豚鼠行ABR检测,第二次ABR检测后对两组豚鼠耳蜗基底膜行鬼笔环肽-异硫氰酸荧光素(Phalloidin-FITC)染色;用硝酸镧透射电镜和常规透射电镜观察两组豚鼠内耳血迷路屏障通透性和紧密连接的变化;Western blot和免疫组织化学染色观察两组豚鼠耳蜗血管纹和螺旋韧带处claudin-5的表达。结果噪声暴露组豚鼠ABR波Ⅲ反应阈较正常对照组阈移40dB以上,差异有统计学意义(P<0.05);基底膜铺片FITC染色示噪声暴露组底回基底膜毛细胞大量缺失,其余部分毛细胞纤毛排列紊乱并出现融合,而对照组毛细胞排列整齐,其纤毛呈V或W型,两组间外毛细胞计数比较差异有统计学意义(P<0.05);透射电镜下,可见噪声暴露组血管内皮细胞间和边缘细胞间的紧密连接均遭到破坏,细胞间隙增大;硝酸镧透射电镜下,大量镧颗粒从血管腔内渗漏到管腔外的组织间隙,而对照组镧颗粒仅局限于血管腔内;Western blot结果示,两组耳蜗外侧壁血管纹均有claudin-5表达,但噪声暴露组的表达量明显低于正常组;免疫组织化学染色也表明,两组豚鼠耳蜗外侧壁毛细血管内皮细胞、边缘细胞等处均有claudin-5阳性表达,但噪声组表达量显著低于正常组(P<0.05)。结论噪声通过下调紧密连接蛋白claudin-5的表达,破坏细胞间紧密连接,增加豚鼠血迷路屏障的通透性,从而导致内耳微环境的破坏,是噪声性聋可能的发病机制之一。