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。     

IL-2、TNF-αmRNA在迷路炎豚鼠耳蜗中的表达

目的探讨迷路炎时,豚鼠耳蜗IL-2、TNF-α mRNA的表达变化及其与迷路炎的关系.方法随机将24只豚鼠分为实验组和对照组,实验组18只、36耳,将1mg.ml-1内毒素(lipopolysaccharide,LPS)0.2ml缓慢注入中耳腔,对照组6只、12耳注入等量生理盐水,观察6h、48h、14d耳蜗的病理变化;用原位杂交技术检测IL-2、TNF-α mRNA在耳蜗的表达,用Tiger 920图像分析软件分析图象.结果(1)耳蜗形态学改变鼓室注药后6h,实验组豚鼠耳蜗的血管纹、螺旋韧带、蜗螺旋轴静脉等即可见充血、水肿、炎细胞浸润,Corti器支持细胞和感觉细胞轻度肿胀、变性;48h时加重;14d时耳蜗各部分结构恢复正常.对照组豚鼠耳蜗各部分结构无改变;(2)IL-2 mRNA的表达对照组耳蜗无IL-2 mRNA表达;鼓室注射LPS后6h,实验组耳蜗的螺旋神经节、螺旋缘呈阳性表达,血管纹、螺旋韧带呈可疑阳性;而48h、14d无表达;(3)TNF-α mRNA的表达对照组耳蜗无TNF-α mRNA表达;鼓室注射LPS后6h,TNF-α mRNA广泛表达于实验组耳蜗的螺旋神经节、Corti器、螺旋缘、血管纹、螺旋韧带等部位,48h表达明显增强(P＜0.01),14d时仅螺旋神经节呈弱阳性染色.结论IL-2、TNF-α可能与急性迷路炎的发生有关,TNF-α则可能起着更为重要的作用.     

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。     

电子自旋共振技术观察急性声损伤后耳蜗自由基改变

目的:应用电子顺磁自旋共振(ESR)技术观察急性声损伤后豚鼠耳蜗自由基的变化规律.方法:将正常白毛红目豚鼠48只分为3组:A组(对照组)取6只豚鼠不给予噪声刺激,分别在检测听功能后测定自由基和硝酸银染色.B组21只豚鼠在(125±1)dB SPL稳态噪声暴露2h后分别于即刻、2h、6h,12h、24h、48h、72h施行ABR测试和ESR检测耳蜗自由基,检测方法为断头后快速取出耳蜗,液氮速冻.样品处理后放入ESR系统谐振腔中检测自由基含量.C组21只豚鼠在噪声暴露后于上述时间点检测听觉功能并取基膜行硝酸银染色观察Corti器毛细胞形态改变.结果:①正常豚鼠耳蜗中有少量自由基存在,其相对自由基值为(21.68±1.27).噪声暴露后即刻取样组自由基值明显升高,在暴露后2h达峰值(147.01±4.95)dB SPL,此后逐渐下降,至72h恢复至接近正常水平(53.12±2.57)dB SPL;②在125dB SPL的急性噪声暴露后,豚鼠的听阈明显提高,至6h达到峰值(73.89±2.41)dB SPL,直至72h仍未恢复到暴露前正常水平(50.28±1.48)dB SPL;③急性声损伤后形态学改变表现为外毛细胞纤毛紊乱、排列不规则,部分区域可见毛细胞缺失.结论:①急性噪声暴露后,豚鼠耳蜗内自由基水平明显增高,并在2h达峰值;②应用ESR技术检测耳蜗组织中自由基含量的方法具有直接、客观和灵敏的特点,ESR技术可用于某些内耳急性损伤动物模型的实验观察.     

压力波对豚鼠耳蜗血管纹、毛细胞心钠素免疫活性改变的研究

目的为探讨压力波的致聋机理,对豚鼠耳蜗血管纹(SV)、毛细胞(HC)中心钠素免疫活性(ANP-IR)的改变及与听阈阈移的相关性进行研究。方法采用免疫细胞化学(ABC法)法、图像分析听性脑干反应测听技术(ABR)对压力波暴露后不同时间分组的豚鼠耳蜗SV、HC中ANP-IR产物进行检测。结果压力波暴露后6、12、24h和48h组SV组织中ANP-IR光密度值较对照组均有明显的差异(P<0．05),其中24h组最高;冲击波暴露后6、12、24h组内毛细胞(IHC)中ANP-IR阳性产物的光密度值较对照组明显增高(P<0．05),其中以12h组最高。二者的变化均与听阈阈移有明显的正相关性(r1=0．8175,P＞0．05;r2=0．9185,P＞0．05)。而外毛细胞(OHC)中ANP-IR阳性产物变化不明显。结论压力波暴露后,SV组织中ANP的增高可能是内耳的一种代偿机制;IHC和OHC中ANP-IR的变化可能是冲击波对其损伤机制的不同表现。     

大鼠耳蜗胶质细胞的分离培养和纯化北大核心CSCD

目的探讨大鼠耳蜗胶质细胞的体外分离培养和纯化的方法,为进一步研究耳蜗胶质细胞功能提供实验基础。方法分离出生后3天的新生大鼠(18只,36耳)耳蜗轴组织,通过组织酶消化及阿糖胞苷补充法、组织酶消化及免疫磁珠分选法、组织块培养及细胞冷喷注法三种方法(各2只,4耳,均重复三次)进行耳蜗胶质细胞分离、培养和纯化,通过细胞计数、CCK-8检测以及胶质细胞标记物免疫荧光染色计算细胞的产量、活性及纯度。结果组织块培养及细胞冷喷注法获得的耳蜗胶质细胞的产量尽管最低,但其细胞活性最高并优于其它两种方法,且细胞纯度较高,接近组织酶消化及免疫磁珠分选法。结论蜗轴组织块培养及细胞冷喷注法获得的耳蜗胶质细胞活性良好且细胞纯度较高,操作简便且成本低,可作为一种便捷的耳蜗胶质细胞体外培养和纯化方法。     

电离辐射对耳蜗毛细胞超微结构的影响

目的 观察电离辐射对耳蜗毛细胞超微结构的损伤情况.方法 将健康豚鼠15只随机分成二组:单纯放射组(单放组)10只,对照组5只,每组观察10耳.对照组不放射,单放组用直线加速器所产生的6Mev电子线对豚鼠的耳颞部予以分次放射(2 Gy/天),总剂量达到60 Gy,行透射电镜及扫描电镜观察两组豚鼠耳蜗毛细胞的形态学变化.结果 透射电镜观察见对照组耳蜗外毛细胞边界清楚,细胞无肿胀,表皮板完整,线粒体嵴完整,核膜完整;单放组耳蜗外毛细胞边界不清,细胞肿胀变形,线粒体空泡变,线粒体嵴断裂,核膜不完整,异染色质增多.扫描电镜观察见对照组耳蜗外毛细胞的纤毛排列整齐无倒伏、缺失;单放组耳蜗外毛细胞的纤毛明显倒伏、缺失、排列紊乱.结论 分割剂量电离辐射对豚鼠耳颞部放射可造成耳蜗毛细胞超微结构损害.     

丹参注射液对庆大霉素耳中毒豚鼠耳蜗一氧化氮生成的影响

目的　研究丹参注射液对庆大霉素 (GM)耳中毒豚鼠耳蜗一氧化氮 (NO)生成的影响 ,探讨丹参注射液对GM耳蜗毒性的防护作用及其作用机制。方法　豚鼠随机分成正常对照组、GM组、GM +丹参组和丹参组 ,应用改良镀铜镉还原法测定豚鼠耳蜗组织中NO含量 ,同时结合听性脑干反应 (ABR)测试 ,观察用药前后听阈变化。结果　GM +丹参组豚鼠耳蜗NO含量和ABR阈值均明显低于GM组 (P <0 .0 1) ;且各组NO含量变化与ABR阈值高度相关 (r正常对照 =0 .96 5 ;rGM=0 .990 ;rGM +丹参 =0 .880 ;r丹参 =0 .980 ;P <0 .0 5 ,P <0 .0 1)。结论　丹参注射液可通过抑制GM引起的NO过量生成 ,以减轻GM的耳毒性损伤 ,从而改善听功能。这可能也是丹参注射液拮抗GM耳蜗毒性的作用机制之一。     

高速率电刺激对豚鼠听神经兴奋性的影响

目的：评估高速率电刺激对豚鼠听神经兴奋性的影响。方法：切开受试豚鼠圆窗膜,标准电极插入鼓阶大约4mm,两个靠近蜗尖的电极作为刺激电极,切开圆窗用压碎的颞肌封住。随机选择一耳为刺激耳,另一耳则为对照耳,均安装上标准电极,刺激电极在整个测试过程中保持不动。在保持刺激强度处在临床正常水平[电刺激诱发听觉脑干电位(EABR)阈值上6dB]情况下,用200(n=14)、400(n=10)、1000(n=11)、2000(n=10)脉冲数／s(PPS)四种不同的电刺激速率急性刺激45只听力正常豚鼠鼓阶内电极2h,记录急性刺激前、后3h内EABR的阈值和Ⅰ波幅值,比较急性刺激前、后EABR的Ⅰ波幅值的变化。结果：急性刺激电流强度固定在临床正常水平(EABR阈上6dB),急性刺激后EABR的Ⅰ波幅值同急性刺激前相比,采用200PPS刺激速率平均约升高20％;400PPS约升高9％;1000PPS约升高7％;2000PPS约升高30％。结论：在刺激电流强度为阈上6dB的情况下,人工耳蜗言语编码策略如果应用1000、2000PPS高速率电刺激不会导致听神经的兴奋性下降。此实验为临床上研制新的人工耳蜗采取高刺激速率语音信号处理方案提供了依据。     

鼠尾胶原在原代培养耳蜗血管纹边缘细胞中的应用

目的：建立利用自制的鼠尾胶原培养大鼠耳蜗边缘细胞的方法。方法：制作鼠尾胶原,并观察利用自制的鼠尾胶原所培养出大鼠耳蜗边缘细胞的效果。结果：自制的鼠尾胶原能正常地培养出大鼠耳蜗边缘细胞,细胞角蛋白18表达阳性,免疫组织化学结果和扫描电镜证实所培养的细胞具有典型的分泌上皮细胞特征。结论：成功建立了利用自制鼠尾胶原培养大鼠耳蜗边缘细胞的方法,并且制作简便,成本低廉。     

Primary culture of vital marginal cells from cochlear explants of the stria vascularis

Summary Explants of the stria vascularis and spiral ligament were dissected from guinea pig cochleae and were successfully cultivated for several weeks. After 2 days, fibroblast-like cells of the spiral ligament covered the bottom of the cell culture dish around the explant. Marginal cells of the stria vascularis proliferated and grew on the luminal surface towards the border of the explant at a rate of 15 m/day. At day 6 in culture the proliferating marginal cells reached the border of the explant and then advanced to the bottom of the cell-culture dish. There the marginal cells replaced fibroblast-like cells and built an epithelial hexagonal-shaped monolayer. Light microscopic and transmission electron microscopic investigations revealed that the cultured cells were viable and that typical morphological characteristics of marginal cells were preserved. Cultivation of these cells provides a unique model for studies of physiological properties of marginal cells of the stria vascularis.     

Migration of cochlear lateral wall cells

The role of apoptosis and proliferation in maintenance of cochlear lateral wall cells was examined. The methods employed for detection of apoptosis were the Hoechst fluorescence stain and TUNEL (TdT-mediated dUTP-biotin nick-end-labeling) assay, and proliferations were 5-bromo-2'-deoxyuridine (BrdU) incorporation and presence of the proliferating cell nuclear antigen. The incidence of apoptosis in the strial marginal cell was 50% greater (32.9+/-3.7%) than strial intermediate and basal cells but similar to spiral ligament cells. Although division of marginal strial cells was rarely detected, a significant number of proliferating cells in the remaining stria vascularis and spiral ligament were observed. These data implied that replacement of marginal cells arose elsewhere and could be followed by a BrdU-deoxythymidine pulse-chase study. At 2 h post injection, nuclear BrdU in marginal cells was not detected; however, by 24 h post injection, 20-25% of marginal cell nuclei were BrdU-positive. These observations are consistent with the hypothesis that marginal cells were replaced by underlying cells. Cell migration appears to be an important mechanism for preserving the function and structure of the stria vascularis.     

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.     

新生小鼠膜迷路耳蜗外侧壁的组织培养

目的培养小鼠耳蜗螺旋韧带/血管纹来源的细胞,为体外研究提供细胞模型。方法显微解剖新生小鼠耳蜗螺旋韧带/血管纹组织,组织块外植培养,胰蛋白酶消化分离细胞,免疫组织化学染色,原位透射电镜观察鉴别细胞的来源。结果自外植耳蜗螺旋韧带/血管纹组织生长出上皮样细胞及成纤维样细胞。前者呈典型的上皮细胞形态,表达细胞角蛋白,并具血管纹边缘细胞的超微结构特征。后者呈成纤维细胞形态,表达波形蛋白,具有耳蜗螺旋韧带成纤维细胞的超微结构特征。结论培养出小鼠耳蜗血管纹边缘细胞及螺旋韧带成纤维细胞来源的原代上皮细胞及传代成纤维细胞,为体外研究提供了细胞模型及方法。     

The volume density of cells and capillaries of the normal stria vascularis

The purpose of this study was to provide morphometric (i.e. quantitative anatomical) data on the normal chinchilla stria vascularis. Five normal chinchillas were used in the present investigation and four regions of the cochlea were examined in each animal. The width, radial area and number of marginal cells across the stria's width increased from the cochlear apex toward the base. The increase in strial width and area appeared to be due to hyperplasia of the marginal cells. The mean total endolymphatic surface area of the stria vascularis was estimated to be 7.4 mm2 (S.E. = 1.23). The mean total volume of the stria vascularis was estimated to be 0.15 microliter (S.E. = 0.01). In addition, using a stereological method we found that the volume density of the cells and capillaries of the stria vascularis was constant along the length of the scala media. The mean (+/- S.E.) volume density of the stria cells and capillaries was estimated to be: marginal cells = 0.528 (0.013), intermediate cells = 0.212 (0.026), basal cells = 0.163 (0.009) and capillaries = 0.097 (0.009).     

豚鼠血管纹缘细胞的原代培养

目的:建立豚鼠内耳血管纹缘细胞的体外培养系统,为研究缘细胞的功能提供良好的材料。方法:采用移植培养技术,将活体分离的血管纹组织块接种于无菌塑料培养皿中,于5％CO2 37℃恒温箱内培养,每周换液2次,观察细胞生长情况。利用免疫组织化学技术,将抗细胞角蛋白抗体和抗波形蛋白抗体用于检测培养细胞。制作透射电镜标本观察培养细胞的超微结构。结果:培养细胞成功生长4周,具有多角形细胞和梭形细胞两种不同形态,免疫组织化学反应显示角蛋白、波形蛋白染色于多角形细胞胞浆中,梭形细胞仅波形蛋白呈阳性反应。透射电镜示多角形细胞具有紧密接合和桥粒等上皮细胞的特征。结论:采用移植块培养技术,成功建立豚鼠内耳血管纹缘细胞的原代培养方法,为研究缘细胞的功能提供了合适的细胞模型。     

Ultrastructure of cultured marginal cells of the guinea pig cochlea.

Explants of stria vascularis and spiral ligament of guinea pig cochlea were kept in primary culture. On the explant, proliferating marginal cells advanced by 15 microns/day, suggesting that in vivo defects of the strial epithelium can be covered by new marginal cells. The marginal cells growing in the cell culture dish had a diameter of 12.8 +/- 0.7 microns and formed an epithelial monolayer. Adjacent cells were connected by desmosomes and tight junctions. The cells were uniformly polarized. The apical membrane had small invaginations and numerous microvillus-like extensions, and the convoluted lateral membrane interdigitated with adjacent cells. The basal infoldings were smaller in cultured cells than in vivo; mitochondria were dispersed in the entire cytoplasm rather than concentrated in basolateral infoldings. The basal membrane infoldings of cultured marginal cells did not interdigitate with underlying fibroblast-like cells. Marginal cells were separated from underlying fibroblast-like cells by a fluid-filled space which was sometimes enlarged, leading to the formation of "domes" in the otherwise planar epithelium.     

Quantitative assessment of the rat stria vascularis

Stria vascularis tissues from standardized regions in the basal, middle and apical turns of the rat cochlear duct were assessed quantitatively. Strial width, number of marginal cells across the strial width, radial area, as well as the volume density of the different components of the stria vascularis were determined for each standardized region. Strial width, number of marginal cells across the strial width and the radial area were greatest in the basal region and least in the apical region of the cochlea. The volume density of intermediate cells and capillary space was statistically unchanged in the three examined regions of the stria vascularis. However, the volume density of marginal cells and that of basal cells were different between regions. The volume density of marginal cells was highest in the basal turn while the volume density of basal cells was greatest in the apical turn. An objective assessment of the response of the stria vascularis to environmental conditions can be made by kant of its cellular architecture, providing a means to compare the effects of various agents between animal models used to study human inner ear dysfunction.