鼠尾胶原在毛细胞膜片钳实验中的应用

目的　探讨应用鼠尾胶原在豚鼠前庭毛细胞膜片钳实验中的促进毛细胞贴壁效果。方法　制作鼠尾胶原 ,观察全细胞膜片钳实验中 ,自制的鼠尾胶原对前庭毛细胞的贴壁黏附作用。结果　无鼠尾胶原时 ,前庭毛细胞悬浮于外液 ,不宜封接 ;有鼠尾胶原时 ,前庭毛细胞贴附于培养皿底壁 ,易于封接和长时间 (约 8h)的观察和记录。鼠尾胶原对前庭毛细胞具有良好的贴壁黏附促进作用。结论　鼠尾胶原能促进毛细胞贴壁 ,涂布鼠尾胶原有利于膜片钳实验的长时程记录 ;并且制作简便 ,成本低廉     

鼠尾胶原在毛细胞膜片钳实验中的应用

目的 探讨应用鼠尾胶原在豚鼠前庭毛细胞膜片钳实验中的促进毛细胞贴壁效果。方法 制作鼠尾胶原，观察全细胞膜片钳实验中，自制的鼠尾胶原对前庭毛细胞的贴壁黏附作用。结果 无鼠尾胶原时，前庭毛细胞悬浮于外液，不宜封接；有鼠尾胶原时，前庭毛细胞贴附于培养皿底壁，易于封接和长时间（约8h)的观察和记录。鼠尾胶原对前庭毛细胞具有良好的贴壁黏附促进作用。结论 鼠尾胶原能促进毛细胞贴壁，涂布鼠尾胶原有利于膜片钳实验的长时程记录；并且制作简便，成本低廉。     

前庭学

20030445鼠尾胶原在毛细胞膜片钳实验中的应用/郭长凯…//中华耳鼻咽喉科杂志一2002,37(4)一307一309 目的:探讨应用鼠尾胶原在豚鼠前庭毛细胞膜片钳实验中的促进毛细胞贴壁效果。方法:制作鼠尾胶原,观察全细胞膜片钳实验中,自制的鼠尾胶原对前庭毛细胞的贴壁勃附作用。结果:无鼠尾胶原时,前庭毛细胞悬于外液,不宜封接;有鼠尾胶原时,前庭毛细胞贴附于培养皿底壁,易于封接和长时间(约sh)的观察和记录。鼠尾胶原对前庭毛细胞具有良好的贴壁豁附促进作用。结论:鼠尾胶原能促进毛细胞贴壁,涂布鼠尾胶原有利于膜片钳实验的长时程记录;并且制作简…     

鼠尾胶原为底物的人鼻腔纤毛上皮细胞培养模式的建立

目的 建立以鼠尾胶原为贴附底物的人鼻腔纤毛上皮细胞的体外培养模式,为人鼻腔黏液纤毛运输系统的研究提供科学有效的方法.方法 制备鼠尾胶原并铺片,以鼠尾胶原为贴附底物组织块培养法培养人鼻腔纤毛上皮细胞,培养7天时进行HE染色及扫描电镜和透射电镜观察细胞形态和结构,应用高速摄像技术测量纤毛摆动频率.结果 鼠尾胶原要平坦均匀,平均厚度约为1 mm;HE染色可见上皮细胞呈单层向周围爬开;扫描电镜下见纤毛上皮细胞呈不规则多角形,纤毛周围可见微绒毛;透射电镜下可见纤毛上皮细胞间为紧密连接;同一细胞任意两点纤毛摆动频率是相同的;同一来源体外培养的钩突和下鼻甲的纤毛摆动频率是相同的.结论 以鼠尾胶原为贴附底物人鼻腔纤毛上皮细胞的体外培养模式的成功建立,为今后研究鼻内用药对纤毛清除功能的影响提供了良好的方法和途径.     

大鼠耳蜗大上皮嵴及小上皮嵴细胞永生细胞系的建立

目的：建立大鼠耳蜗大、小上皮嵴细胞永生细胞系。方法：取P1大鼠耳蜗,分离出耳蜗上皮薄片组织,并与含SV40-LT抗原的逆转录病毒共培养,从而实现细胞的永生化。利用单克隆法纯化细胞系,并通过形态学、免疫组织化学等方法对细胞系进行鉴定。结果：大鼠耳蜗大、小上皮嵴细胞永生细胞系基本保持了细胞原代的表型特征：呈现上皮细胞样的多角形外观;不同世代的细胞均表达Isletl及SV40-LT抗原。该系目前已培养3个月,超过20代,传代时间为3～4d,传代比例为1：5。结论：本实验成功建立了大鼠耳蜗大、小上皮嵴细胞的永生细胞系,这为大、小上皮嵴细胞生物学特性及相关功能,特别是毛细胞再生及前体细胞移植治疗感音神经性聋方面的研究奠定了坚实的实验基础。     

pEGFP-N1/MnSOD重组质粒的构建及体外表达

目的:构建大鼠源性锰超氧化物歧化酶(MnSOD)基因真核表达载体pEGFP-N1/MnSOD,并检测其在原代培养的大鼠耳蜗血管纹边缘细胞中的表达。方法:从大鼠心肌组织中扩增出SOD基因,克隆入融合表达载体pEGFP-N1中,用脂质体法转染大鼠耳蜗血管纹边缘细胞,激光共聚焦显微镜观察绿色荧光蛋白及目的基因的表达,流式细胞仪检测转染效率。结果:构建了大鼠源性MnSOD基因真核表达载体pEGFP-N1/MnSOD,并观察到转染/感染48h后,绿色荧光蛋白及目的基因在大鼠耳蜗血管纹边缘细胞中表达,目的基因的转染效率为23.47%。结论:成功构建了大鼠源性MnSOD基因真核表达载体pEGFP-N1/MnSOD,并使目的基因成功在大鼠耳蜗血管纹边缘细胞中表达,为内耳抗氧化基因治疗奠定了基础。     

大鼠耳蜗大上皮嵴的分离和鉴定

目的：探讨利用酶消化和机械解剖相结合分离大鼠耳蜗大上皮嵴(GER)的方法。方法：取出生后0～3d的SD大鼠耳蜗基底膜后,将其放人含有嗜热菌蛋白酶和少许DNA酶的D-Hank溶液中,37℃孵箱孵育25min进行酶消化,然后在高倍显微镜下进行显微机械分离;对分离的GER细胞用免疫组织化学和RT-PCR扩增GER细胞特异性表达基因Hesl的方法进行鉴定,并进行GER的原代培养以验证其活性。结果：免疫组织化学染色表明分离的GER细胞是单一的上皮组织;对Hesl基因进行RT-PCR分析表明,分离的GER细胞表达该基因;分离的GER具有活性并可以进行原代培养。结论：利用酶消化和机械解剖相结合的方法可以分离出纯粹的有活性的GER,为进一步建立GER细胞系,进行各种基因转染试验以及耳蜗内的各种基础实验建立细胞模型提供有效的实验手段。     

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。     

听力学及言语疾病杂志

新生大鼠耳蜗Corti器体外培养及其转基因表达刘英鹏,鄢开胜,王建亭,龚树生听力学及言语疾病杂志,2006,14(2):127-129目的:建立新生大鼠耳蜗Corti器体外培养模型及观察外源基因表达。方法:采用出生后1～3天龄大鼠,分离出的耳蜗Corti器组织置入表面覆盖有胶原凝胶的培养皿中,在含     

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。     

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。     

Effect of a beta-stimulant on the inner ear stria vascularis

In vivo and in vitro experiments were conducted to investigate the effect of alpha-isoproterenol on the inner ear stria vascularis with intracellular cytochrome oxidase activity used as an index. Intraperitoneal injection of alpha-isoproterenol (5 mg/kg) was performed in 10 rats, and that of physiological saline in 4 rats, for 21 consecutive days. After the 3-week treatment, bilateral cochleas were excised for frozen sections and stained for cytochrome oxidase. The staining density of the stria vascularis for the enzyme was analyzed with a computer. Electron microscopic observation was also performed for some specimens. As for the in vitro experiments, bilateral cochleas from 6 normal rats were excised for cell culture. Cochlear cells from the right ear were cultured with medium containing alpha-isoproterenol (10-micromol/L concentration), and those from the left ear with medium alone. After 3-day culture, the enzyme activity of cytochrome oxidase in the stria vascularis was quantified by the same method used for the in vivo experiments. Cytochrome oxidase activity was markedly elevated in the alpha-isoproterenol group. The activity tended to be higher in the lower turns of the cochlea. Electron microscopy revealed that numerous mitochondria were present in marginal cells that protruded into the endolymphatic space. The enzyme activity was also elevated in the stria vascularis from cochlear specimens in the alpha-isoproterenol group of the in vitro experiment. The above results suggest that alpha-isoproterenol accelerated the metabolic activity of the cells that constitute the stria vascularis. The increase in activity was probably attributable to direct pharmaceutical effects of the beta-stimulant, rather than an increase in blood flow. It is possible that the cells that constitute the stria vascularis may have beta-receptors.     

Differentiation of cyst-forming stria vascularis tissues in vitro

The marginal cells of the stria vascularis possess distinctive morphological characteristics associated with their role in endolymph production. Interestingly, when stria-derived epithelial cells are grown in association with the underlying mesenchyme, the final differentiation of these cell types does not occur. Beyond the rudimentary polarity that is established, similar to that shown in epithelial monolayers, cells in culture bear only a slight resemblance to their marginal cell counterparts in vivo. The ultrastructural features that typify these epithelia, extensive cytoplasmic invaginations, with an abundance of mitochondria, and darkly stained cytoplasm, are not evident under standard culture conditions. In order to determine whether fluid transport, a key function of the stria vascularis, has an effect on the ultrastructural morphology, we examined de novo stria vascularis tissues that formed a fluid-filled cyst in vitro. We found that only cells associated with the luminal structure demonstrated dark cytoplasmic staining and amplification of the basolateral membrane of the marginal cells. Additionally, other epithelial features, such as mitochondria-rich and microvilli-rich cells, were observed in cyst-forming tissues. The enhancement of the marginal cell specializations was not as robust as that observed in vivo; however, they were clearly more extensive when compared to cells in the same culture that were not associated with a fluid-filled lumen. Thus it appears that fluid transport may be necessary to maximize differentiation of stria vascularis tissues in vitro.     

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。     

Primary culture of strial marginal cells of guinea pig cochlea: growth, morphologic features, and characterization.

To further investigate the cellular mechanisms involved in the formation of endolymph, primary cultures of marginal cells of guinea pig were established. Minute explants obtained by mechanical dissociation of stria vascularis were plated on collagen type I precoated impermeable substrate in serum-free, hormone-supplemented medium. A confluent layer of epithelial-like cells was obtained within 2 weeks. The cultured cells formed domes, demonstrating that they retain some of their transepithelial properties. Polarization was also suggested by electron microscopic observation of apical microvilli and tight junctions. Immunohistochemical methods revealed that the cultured cells coexpressed cytokeratin and vimentin, demonstrating their epithelial origin, although some degree of dedifferentiation occurred. Thus, a primary culture of marginal cells can be established that may be a suitable model for an in-depth investigation of the function of the marginal cells.     

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.     

Characterization of marginal and Claudius' cells growing from cochlear explants in vitro.

Tissue specimens of stria vascularis together with spiral ligament were transferred from the guinea pig cochlea to tissue culture dishes. To characterize and identify cells growing out from the explants, indirect immunofluorescence microscopy was used. The expression of the intermediate-sized filaments vimentin and cytokeratin 18 in cells on the surface of tissue specimens and in cells growing out from the explants after different cultivation periods were compared. Basically, three types of cells grew from the explants during several days: marginal cells, Claudius' cells and fibroblast-like cells. In primary cultures of explants, growth of marginal cells was observed in 25% of the dishes. Their proliferative activity, estimated by the use of the BrdUrd-DNA antibody, started in the stria vascularis and continued across the attachment of Reissner's membrane down to the bottom of the cell culture dish. The newly-formed marginal cells expressed cytokeratin 18 in the same way that original marginal cells on the tissue specimen do. If the newly-formed marginal cells were in contact with fibroblast-like cells or were forming groups (domes) on the bottom, they expressed vimentin. In 3% of the dishes growth of Claudius' cells was observed. Proliferative activity of these cells was found at the point where the basilar membrane was attached to the spiral ligament. New Claudius' cells spread at the opposite side of an explant when compared with the location of new marginal cells. Original as well as newly-formed Claudius' cells contained cytokeratin 18. Fibroblast-like cells were commonly present in cultures and contained only vimentin.     

胎儿耳蜗血管纹的扫描电镜观察

目的：借助扫描电镜技术观察血管纹的表面结构,以便对血管纹的结构和功能提供新的信息。方法：标本取自尸检3个足月胎儿的6个颢骨,在死后尽可能快速取出耳蜗,经处理后,借助扫描电镜技术观察人胎儿耳蜗血管纹的超微结构。结果：借助扫描电镜技术可从耳蜗基底圈到顶圈,上起前庭膜嵴,下到基底膜的基底嵴全面观察血管纹的各个部分,血管纹边缘细胞表面结构呈圆球形．细胞表面有许多微绒毛。螺旋凸部位有一个细胞移行区,这个区域的边缘细胞表面形态明显不同,细胞细K或呈不规则的多角形细胞,细胞边界微绒毛丰富,显出明显的细胞界限,细胞表面分布均匀的微绒毛。血管纹断面的观察还可获得中间细胞、基底细胞和毛细血管结构。结论：扫描电镜观察胎儿耳蜗血管纹,可获得整个血管纹全貌的表面精细结构,尤其是边缘细胞的表面形态,通过对血管纹断面的观察还可获得中间细胞、基底细胞的精细结构特征,为认识血管纹的结构和功能提供新的信息。     

Establishment of inner ear epithelial cell culture: isolation, growth and characterization

Select epithelial regions of the bovine inner ear were established and maintained in cell culture. Marginal cells from the stria vascularis and dark cells from the posterior wall of the utricle were isolated, dissociated and placed in culture medium. Within 24 h, cellular islands of hexagonal-shaped, epithelial-like cells from both the stria vascularis and posterior utricular wall were readily identifiable by inverted light microscopy. Ultrastructural examination of both the cultured stria marginal cells and utricular dark cells revealed that both cell types had numerous microvilli on their apical surfaces and interdigitating infoldings of their basolateral surfaces. Apical tight junctional complexes were present between apposing cells. These findings demonstrate that inner ear bovine epithelial cells can be successfully isolated and maintained in culture, and that such cells retain certain of their in vivo morphological characteristics.