庆大霉素对豚鼠内淋巴囊和血管纹Na^＋—K^＋—ATP酶活性的影响

应用酶细胞化学技术和计算机图像分析，对豚鼠连续肌注庆大霉素２－１５ｄ后内淋巴囊上皮细胞和血管纹边缘细胞的Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶活性变化进行了定量观察。结果发现，与正常对照组相比，用药２ｄ后两类细胞的Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶活性反应产物没有明显变化，但连续用药５ｄ后二者的产物均明显减少，１０ｄ和１５ｄ仍呈继续轻微减少趋势。表明庆大霉素既可抑制血管纹边缘细胞Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶活性，降低其     

庆大霉素对豚鼠内淋巴囊和血管纹Na~＋－K~＋－ATP酶活性的影响

应用酶细胞化学技术和计算机图像分析，对豚鼠连续肌注庆大霉素（１００ｍｇ·ｋｇ－１／ｄ）２～１５ｄ后内淋巴囊上皮细胞和血管纹边缘细胞的Ｎａ＋－Ｋ＋－ＡＴＰ酶活性变化进行了定量观察。结果发现，与正常对照组相比，用药２ｄ后两类细胞的Ｎａ＋－Ｋ＋－ＡＴＰ酶活性反应产物没有明显变化，但连续用药５ｄ后二者的产物均明显减少，１０ｄ和１５ｄ仍呈继续轻微减少趋势。表明庆大霉素既可抑制血管纹边缘细胞Ｎａ＋－Ｋ＋－ＡＴＰ酶活性，降低其分泌功能；同时也能抑制内淋巴囊上皮细胞的该酶活性，减弱其液体重吸收功能。     

豚鼠内淋巴囊上皮细胞Na^＋,K^＋—ATP酶不同？…

目的 进一步研究同肉淋巴囊上皮细胞Ｎａ＾＋，Ｋ＾＋－ＡＴＰ酶不同β亚基的表达。方法 采用豚鼠内淋巴囊冰冻切片、原位杂交的方法检测Ｎａ＾＋，Ｋ＾＋－ＡＴＰ酶不同β亚基ｍＲＮＡ在淋巴囊上皮细胞中的表达。结果 在内淋巴囊上皮细胞胞浆内可见Ｎａ＾＋，Ｋ＾－ＡＴＰ酶不同β亚基的阳性颗粒，β１亚基表达较弱，β２亚基表达较强。结论 结合其他报道，内淋巴囊上皮细胞具有多种亚基异构体组成的Ｎａ＾＋，Ｋ＾＋－ＡＴＰ酶     

Na~＋－K~＋－ATP酶活性在豚鼠内淋巴囊的细胞化学定位

Ｎａ￣＋－Ｋ￣＋－ＡＴＰ酶在Ｎａ￣＋－Ｋ￣＋离子主动性跨上皮转运过程中起重要作用。应用对硝基苯酚磷酸（ｐ－ＮＰＰ）为底物的柠檬酸铅一步法Ｋ￣＋－ＮＰＰ酶电镜细胞化学技术，观察了反映Ｎａ￣＋－Ｋ￣＋－ＡＴＰ酶活性的Ｋ￣＋－ＮＰＰ酶在豚鼠内淋巴囊的超微结构定位分布。结果：在透射电镜下观察，内淋巴囊超薄切片上的Ｋ￣＋－ＮＰＰ酶活性反应产物仅分布于上皮细胞底侧面胞膜的胞浆一侧，向上一直延续至上皮细胞间的紧密连接水平，内淋巴腔面胞膜未见反应产物。提示丰富的Ｎａ￣＋－Ｋ￣＋－ＡＴＰ酶活性特征性地分布在内淋巴囊上皮细胞底侧面胞膜，表明Ｎａ￣＋－Ｋ￣＋－ＡＴＰ酶在内淋巴囊上皮Ｎａ￣＋－Ｋ￣＋离子交换过程中的重要地位。     

肾上腺素α2受体在豚鼠血管纹的定位定量研究

用竞争蛋白结合分析法及放射自显影法对豚鼠血管纹肾上腺素α２受体进行了定性定位定量测定，结果显示：加入去甲肾上腺素组（Ａ组）中ｃＡＭＰ量仅为１．３８９±０．８５２ｐｍｏｌ／４０μｌ，较对照组（Ｃ组）７．７００８±０．８６７ｐｍｏｌ／４０μ显著低（Ｐ＜０．０５）；先加入育亨宾再加去甲肾上腺素组（Ｂ组）中ｃＡＭＰ量又复回升达６．９５４±０．９６２（Ｐ＜０．０５），但仍较对照组低（Ｐ＜０．０５）。在经３Ｈ－育亨宾孵育的切片中，血管纹中下部银粒数为９６±１４个／视野，呈条带状分布，与血管纹走向一致，血管纹之外的组织银粒散在均匀，银粒数为２８±７／视野。经细胞立体计量法计算，血管纹中α２受体的含量大约为１．２５×１０９个／ｍｍ３。作者认为，豚鼠血管纹边缘细胞存在α２受体，并推想该受体是通过膜受体－ｃＡＭＰ途径影响细胞膜上Ｎａ＋－Ｋ＋－ＡＴＰ酶的活性，从而参与内淋巴的平衡。     

Na^＋—D^＋—ATP酶活性在豚鼠内淋巴囊的细胞化学定位

Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶在Ｎａ＾＋－Ｋ＾＋离子主动性跨上皮转运过程中起重要作用。应用对硝基苯酚磷酸（ｐ－ＮＰＰ）为底物的柠檬酸铅一步法Ｋ＾＋－ＮＰＰ酶电细胞化学技术，观察了反映Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶活性的Ｋ＾＋－ＮＰＰ酶在豚鼠内淋巴囊的超微结构定位分布。结果：在透射电镜下观察，内淋巴囊超薄切片上的Ｄ＾＋－ＮＰＰ酶活性反应产物仅分布于上皮细胞底侧面胞膜的胞浆一侧，向上一直延续至上皮细胞间的紧密     

鼓阶电刺激后螺旋神经节神经元Na~＋－K~＋－ATP酶和耳蜗传出神经AChE实

用０．１～１．０ｍＡ电流刺激卡那霉素致聋豚鼠耳蜗３小时。三个月后，经组织学处理用积分吸光度定量测定螺旋神经节神经元（ＳＧＮ）Ｎａ￣＋－Ｋ￣＋－ＡＴＰ酶和耳蜗传出纤维乙酰胆碱酯酶（ＡＣｈＥ）。实验结果表明０．１ｍＡ级可增强上述酶的活性；０．４ｍＡ级酶活性无改变；１．０ｍＡ级可显著抑制酶的活性。认为０．１～０．４ｍＡ级电流对豚鼠耳蜗是安全临界域，在人工耳蜗植入电路设计时，有参考意义。     

鼓阶电刺激后螺旋神经节神经元Na^＋—K^＋—ATP酶和耳蜗传出神…

用０．１～１．０ｍＡ电流刺激卡那霉素致聋豚鼠耳蜗３小时。三个月后，经组织学处理用积分吸光度定量测定螺旋神经节神经元（ＳＧＮ）Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶和耳蜗传出纤维乙酰胆碱酯酶（ＡＣｈＥ）。实验结果表明０．１ｍＡ级可增强上述酶的活性；０．４ｍＡ级酶活性无改变；１．０ｍＡ级可显著抑制酶的活性。认为０．１～０．４ｍＡ级电流对豚鼠耳蜗是安全临界域，在人工耳蜗植入电路设计时，有参考意义。     

豚鼠内耳Na,K-ATP酶α亚基异构体的表达

目的 研究豚鼠内耳组织中Na，K-ATP酶α亚基三种异构体αl、α2、α3的表达及其意义。方法 用小鼠抗大鼠Na，K-ATP酶α亚基异构体特异性单克隆抗体，采用免疫组化SP法观察豚鼠耳蜗、半规管、椭圆囊及球囊组织中Na，K—ATP酶α亚基三种异构体的表达模式。结果 αl亚基异构体广泛分布于内耳组织的各个部位，特别是上皮细胞和螺旋神经节细胞中，α2和α3亚基异构体则主要分布于螺旋神经节细胞、Corti器及血管纹。结论 Na，K—ATP酶亚基异构体在内耳不同部位的表达差异表明不同的内耳细胞转运Na^ 和K^ 的能力不同，它们共同作用参与保持内耳内环境的稳定。     

自身免疫性感音神经性聋的内耳代谢与电生理研究

为探讨自身免疫性感音神经性聋（ＡＳＨＬ）的内耳病理生理学机制，采用听觉电生理技术和酶组织化学方法，观察ＡＳＨＬ模型动物的内耳生理功能与组织内主要酶活性的变化。结果示：听视经复合动作电位和耳蜗微音器电位阈值明显升高，内淋巴电位（包括负相）幅值均有不同程度的降低，并与血管纹和内淋巴囊局部组织内Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶和琥珀酸脱氢酶活性改变之间的相关性。表明自身免疫性内耳损伤，进而造成组织内相关酶代谢     

Expression of alpha and beta subunit isoforms of Na, K-ATPase in the guinea pig endolymphatic sac]

OBJECTIVE: To investigate the expression and significance of alpha and beta subunit isoforms of Na, K-ATPase in the guinea pig endolymphatic sac. METHODS: The distribution of alpha and beta subunit isoforms of Na, K-ATPase in endolymphatic sac of guinea pig was identified by immunocytochemistry and in situ hybridization. RESULTS: The expression of Na, K-ATPase alpha and beta subunit isoforms varied among different cell regions of the endolymphatic sac. Epithelial cells of the endolymphatic sac were observed to contain the alpha 1, beta 1 and beta 2 subunit isoforms, and the expression of beta 2 was stronger than that of beta 1. Subepithelial cells contained alpha 1 and alpha 2 subunit isoforms and the expression of alpha 1 was stronger than that of alpha 2. No expression of alpha 3 subunit isoform was observed in endolymphatic sac. CONCLUSION: Na, K-ATPase of endolymphatic sac consists of different alpha and beta subunit isoforms which, working in concert, serve to maintain homeostasis in inner ear.     

庆大霉素对豚鼠血管纹黑色素的影响及其机制

OBJECTIVE: To investigate the effect and its mechanism of gentamicin(GM) on melanin in stria vascularis of guinea pig. METHODS: The differences of auditory thresholds between pigmented and albino guinea pigs, given GM of 150 mg/kg for 7 days, were studied. Moreover, the content of melanosomes, activity of tyrosinase and expression of proliferating cell nuclear antigen(PCNA) in intermediate cells of stria vascularis in gentamicin-treated pigmented guinea pigs were compared with those in control animals by electron microscope and immunohistochemistry, respectively. RESULTS: After gentamicin exposure, the auditory thresholds of all animals increased significantly (P < 0.001), whereas threshold shifts averaged across all frequencies of pigmented animals were much less than those of the albinos(P < 0.001). The number of melanosomes of each examined area (300 microns 2) in intermediate cells was obviously increased from 19.83 +/- 2.74 to 58.33 +/- 16.22. The ratio of tyrosinase reaction products area to the total measured area was significantly increased from 1.65% +/- 0.40% to 3.45% +/- 0.41% after gentamicin exposure. However, the numbers of positive intermediate cells expressing PCNA were 14.08 +/- 2.76 and 13.58 +/- 2.09 before and after gentamicin treatment, respectively. But there was no statistically significant difference between them (P > 0.05). CONCLUSION: The increase of content of melanin in stria vascularis after GM exposure does not result from the change of proliferating activity of melanocytes, but from the enhanced tyrosinase activity. Melanins in stria vascularis may possess the ability to protect the inner ear from ototoxicity of gentamicin.     

Status of cochlear Na,K-ATPase in the aged SHR rat and its possible role in hearing loss

The relationship between levels of Na,K-AT-Pase isoforms was studied in the lateral walls of the cochlea in aged spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Dense staining for the enzyme's α₁ subunit was found especially in the basal turns of the cochlea, while external sulcal cells were more intensely stained in the more apical turns in both SHR and WKY rats. In contrast, staining for β₁ Na, K-ATPase was demonstrable in significant levels in the stria vascularis and suprastrial regions of the SHR rat, with involvement of the basilar suprastrial region pronounced in both strains of animals. Findings suggest a spatially defined, age-induced alteration in cochlear homeostasis with a possible consequent effect on sound perception.     

正常豚鼠内耳水通道蛋白的表达及意义

目的：检测正常豚鼠内耳组织中水通道蛋白（aquaporins,AQPs）的表达,探讨其在内耳液体平衡中的意义.方法：用免疫组织化学方法,以兔抗大鼠AQP0、1、2、3、5、7、8的多克隆抗体,检测正常豚鼠内耳组织中水通道蛋白亚型0、1、2、3、5、7、8的表达.结果：水通道蛋白亚型0、1、2、3、5、7、8在豚鼠内耳有不同程度、不同模式的表达,其中AQP0仅在血管纹上皮细胞、螺旋神经节细胞有较弱的表达,AQP1的分布见于包绕骨迷路、内淋巴囊、内淋巴管的纤维细胞,基底膜鼓阶面细胞、螺旋韧带纤维细胞、螺旋缘纤维细胞、Corti器、内外螺旋沟、血管纹、椭圆囊壁、球囊壁、螺旋神经节细胞等.AQP2表达在血管纹、Corti器、螺旋神经节细胞和内淋巴囊中.AQP3、7、8的分布类似,在螺旋神经节和包绕膜迷路的组织中均有表达,其中Corti器、内外螺旋沟、血管纹、螺旋神经节表达较强,在螺旋韧带、螺旋缘纤维细胞表达较弱.AQP5则在Corti器、内外螺旋沟、螺旋神经节细胞表达较强,在螺旋韧带纤维细胞表达稍弱.结论：在正常豚鼠内耳中,尤其是膜迷路中有多种水通道蛋白亚型,以不同的方式表达,他们可能在维持膜迷路液体平衡中起着协同作用.     

Spiral ligament and stria vascularis changes in cochlear otosclerosis: effect on hearing level.

OBJECTIVE: To investigate the effect of changes within the spiral ligament and stria vascularis on hearing in cochlear otosclerosis, we examined spiral ligament hyalinization, stria vascularis atrophy, and sensory hearing loss in cochlear otosclerosis and described changes in ion transport molecule expression. STUDY DESIGN: Retrospective. SETTING: Tertiary referral center. PATIENTS: Thirty-two cochleae from 24 temporal bone donors with histologic evidence of cochlear otosclerosis, including spiral ligament hyalinization. INTERVENTION: Audiography. MAIN OUTCOME MEASURES: Measurements of spiral ligament width, stria vascularis, and bone-conduction thresholds were compared by the amount of hyalinization. Expression of the ion transport molecules Na,K-ATPase, connexin 26, and carbonic anhydrase II were assessed by immunohistochemical techniques. RESULTS: Hyalinization most often involved the posterior basal turn (88%) and the posterior middle turn (27%). Spiral ligament hyalinization correlated significantly with stria vascularis atrophy in the posterior middle turn of the cochlea (rho = -0.63, p < 0.01). There was a trend toward a significant association in the posterior basal turn (rho = -0.31, p < 0.08). Bone-conduction thresholds at 2,000 and 4,000 Hz were significantly associated with the amount of stria vascularis atrophy (rho = -0.44, -0.40, p < 0.05). In addition, we observed decreased immunostaining for both carbonic anhydrase II with Type I fibrocytes and Na,K-ATPase with stria vascularis and Type II and Type IV fibrocytes of the spiral ligament in cochlear otosclerosis sections compared with normal cochlea. Na,K-ATPase staining within the stria vascularis was further decreased in the presence of spiral ligament hyalinization. No significant differences were seen with connexin 26 immunostaining. However, immunostaining results were somewhat inconsistent. CONCLUSION: These data suggest that spiral ligament structure and function are essential for stria vascularis survival. In addition, dampened expression of ion transport molecules within the spiral ligament and stria vascularis may disrupt potassium ion recycling, resulting in loss of endocochlear potential and sensory hearing loss.     

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。     

小鼠耳蜗血管纹Na-K-2Cl联合转运子1在顺铂耳毒性发生时的改变

目的研究顺铂(cis-dichlorodiammine platinum,Cisplatin)耳毒性发生后耳蜗血管纹Na-K-2Cl联合转运子1(NKCC1)的表达情况,并初步探讨其机制。方法选取健康CBA/CaJ小鼠20只,随机分为对照组和实验组各10只,实验组动物连续腹腔注射顺铂3.5mg.kg-1.d-1,建立顺铂耳毒性小鼠模型,对照组注射等量生理盐水。以听性脑干反应(ABR)阈值作为评价听功能的指标,检测给药前后小鼠听功能的改变,并采用免疫组织化学(SP法)结合免疫荧光实验技术,观察对照组和实验组小鼠腹腔注射顺铂前后耳蜗血管纹NKCC1表达的变化。结果NKCC1在小鼠耳蜗血管纹主要表达于边缘细胞,而顺铂作用后血管纹边缘细胞的NKCC1表达明显减弱,图像分析显示两组平均灰度值差异有显著统计学意义(P<0.01)。结论小鼠顺铂耳毒性作用后血管纹边缘细胞NKCC1的表达量明显减弱,这可能是顺铂耳毒性发生机制中的一个重要环节。     

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...     

速尿对离体培养豚鼠血管纹毒性作用的观察

目的：观察速尿对离体培养的豚鼠血管纹组织的影响，探讨速尿耳毒性的作用机制。方法：20只花色豚鼠随机分成二组：速尿组（n=16），正常对照组（n=4）。应用组织块培养的方法，将血管纹组织培养24小时，随即对不同的实验组分别应用不同终浓度的的速尿（60、300、600、1250、2500μg/ml），分别继续培养30分钟和90分钟，观察培养的血管纹组织学结构。结果：速尿组在组织学方面均未出现血管纹水肿、缘细胞胞浆肿胀、细胞间隙扩大和中间细胞皱缩等病理改变，与对照组相比较血管纹结构无显著性差异。结论：速尿对体外培养的豚鼠血管纹组织无明显诱导水肿的作用，提示速尿耳毒性的产生，可能是间接的作用机制。