大鼠椭圆囊感觉上皮细胞的原代培养及其意义

目的建立大鼠椭圆囊感觉上皮细胞（utricular sensory epithelial cell，USEC）的原代培养体系，为研究毛细胞再生机制提供大量来源一致的细胞。方法取出生1天（postnatal dayl，P1）大鼠的椭圆囊感觉上皮，嗜热菌蛋白酶（thermolysin）消化处理，获得纯椭圆囊感觉上皮。再采用消化法进行原代培养，培养基为DMEM。从第2天开始每日用倒置显微镜观察、照相，对USEC的形态、生长特征进行观察；透射电镜观察；应用细胞角蛋白18及波形蛋白免疫细胞化学方法鉴定USEC来源：应用免疫细胞化学及RT—PCR分别检测毛细胞的特征性标志物calretinin、Brn3．a和AChR α9、myosin Ⅶa mRNA的表达。结果原代培养USEC呈扁平、多角形、核大而圆的上皮细胞形态。细胞之间连接紧密，形成单层时呈“铺路石样”外观，可见由成百个USEC包绕液体而成的dome。原代USEC表达细胞角蛋白，不表达波形蛋白，微绒毛丰富，细胞间连接紧密，提示其上皮起源；表达毛细胞的特征性标志物Bin3．a、calretinin及AchR α9、myosin Ⅶa mRNA，表明培养USEC具有毛细胞前体细胞的特征。结论原代培养的USEC表达毛细胞的特征性标志物，具有毛细胞前体细胞的特征。USEC原代培养体系的建立。为进一步探讨毛细胞再生的机制提供充足的细胞来源。     

应用嗜热菌蛋白酶快速分离活性纯椭圆囊感觉上皮细胞及其意义

目的建立纯椭圆囊感觉上皮细胞(pure utricular sensory epithelial cell,PUSEC)的快速分离方法,为内耳细胞培养及毛细胞再生等机制研究提供大量的活性细胞。方法通过对出生5天大鼠PUSEC的形态、生长特征的观察,采用透射电镜,紧密联接蛋白(ZO-1)、细胞角蛋白、波形蛋白免疫荧光细胞化学、RT-PCR检测毛细胞的特征性标志物Calretinin、Brn3.a mRNA的表达等方法鉴定PUSEC来源。结果PUSEC呈扁平、多角形、核大而圆的上皮细胞形态,细胞之间连接紧密,形成单层时呈“铺路石样”外观。可见由成百个PUSEC包绕液体而成的dome。PUSEC表达ZO-1和细胞角蛋白,不表达波形蛋白,表达毛细胞的特征性标志物Calretinin、Brn3.a的mRNA。结论PUSEC来源于椭圆囊感觉上皮;纯椭圆囊感觉上皮细胞成功分离为内耳细胞培养及毛细胞再生机制的研究提供大量的活性细胞;成功分离的关键成份是嗜热菌蛋白酶。     

大鼠椭圆囊再生毛细胞前体细胞来源的初步研究

目的 探讨哺乳动物椭圆囊再生毛细胞前体细胞的可能来源.方法 取出生1天的大鼠的椭圆囊,经嗜热菌蛋白酶(thermolysin)处理,消化法进行原代培养.在倒置显微镜下观察椭圆囊感觉上皮细胞(utricular sensory epichelial cell,USEC)的形态、生长特征;透射电镜观察、免疫细胞化学法检测上皮细胞角蛋白18、波形蛋白等鉴定USEC上皮细胞来源.免疫细胞化学法、RT-PCR技术检测支持细胞标记物p27k1plmRNA及毛细胞的特征性标记物Brn3a、Calretinin及AchRa9、Myosin Ⅶ a mRNA的表达.结果 原代培养的USEC呈扁平、多角形、核大而圆的上皮细胞形态,细胞之间连接紧密,形成单层时呈"铺路石样"外观.可见由数百个USEC包绕液体而成的dome(穹窿样)结构,表达细胞角蛋白,不表达波形蛋白,微绒毛丰富,细胞间连接紧密,提示其上皮来源.原代培养的USEC表达支持细胞标记物p27k1pl mRNA及毛细胞的特征性标志物Brn3a、Calretinin及AchRa9、MyosinⅦa mRNA,表明培养的USEC可能来源于支持细胞并具有毛细胞的特性.结论 原代培养的USEC能产生毛细胞样细胞且表达支持细胞标记物,表明其来源为支持细胞.椭圆囊感觉上皮的支持细胞可能为毛细胞再生的前体细胞之一.     

大鼠耳蜗感觉上皮细胞的原代培养及其意义

目的：建立大鼠耳蜗感觉上皮细胞（CSEC）的原代培养体系，为研究毛细胞再生的分子机制提供大量来源一致的细胞。方法：取出生1d（P1）的大鼠耳蜗感觉上皮（Corti器）作组织块培养，选择性消化抑制成纤维样细胞（FLC）生长；应用有限稀释法纯化CSEC，倒置显微镜下观察CSEC的形态、生长特征；应用细胞角蛋白18、波形蛋白、Brn3．a和Calretinin的免疫细胞化学及透射电镜等进行鉴定。Brdu标记CSEC核DNA观察CSEC有丝分裂活动。结果：培养第2天CSEC从组织块迁移出来，呈扁平、多角形、核大而圆的上皮细胞形态，细胞之间连接紧密，形成单层时呈“铺路石样”外观；FLC呈梭形“鱼群样”包绕CSEC生长，增殖速度快于CSEC。由纯化的CSEC构成的上皮单层可见由成百个CSEC包绕液体形成的dome。CSEC表达毛细胞的特征性标志物，证实培养的CSEC具有毛细胞前体细胞的特征；并通过有丝分裂活动产生新的细胞即毛细胞样细胞。结论：体外培养的CSEC表达毛细胞特征性标志物，具有毛细胞前体细胞的特征。CSEC原代培养体系的建立，为进一步探讨毛细胞再生的分子机制提供了实验条件。     

小鼠耳蜗感觉上皮细胞的自然培养诱导毛细胞的产生

目的 培养小鼠耳蜗上皮细胞,寻找听觉毛细胞的前体细胞,从而研究听觉毛细胞的再生。方法 改良细胞培养基和培养技术,建立小鼠耳蜗听觉上皮细胞的培养;用免疫细胞化学方法和BrdU标记法检测培养细胞的性质和分裂状态。结果 培养的听觉上皮细胞表现为大而扁平的上皮细胞形态,并且表达上皮细胞的标志F-actin和cytokeratin,部分新生的细胞可被早期毛细胞的特异标志calretinin着染,表明有听觉毛细胞样的细胞产生,这种现象经3次传代培养后仍然存在。结论 自然细胞培养方法可能诱导小鼠听觉毛细胞的产生,在小鼠的耳蜗内可能存在听觉毛细胞的前体细胞,而这些前体细胞是否是组织特异性干细胞还需要更进一步的研究。     

离体培养时Wnt3a对椭圆囊毛细胞再生的影响

目的 在小鼠椭圆囊体外培养模型上,通过Wnt3a激活经典WNT信号,与DMSO共同作用,研究其对椭圆囊毛细胞再生的影响。方法 40只小鼠随机分成8组。实验第一部分：小鼠椭圆囊经4mmol/L新霉素处理后,在不同浓度(0、25、100、200ng/mL)Wnt3a的培养液中继续培养1d,培养结束后对β-catenin,毛细胞纤毛及细胞核进行免疫荧光染色。实验第二部分：小鼠椭圆囊经4mmol/L新霉素处理后,在含25ng/mL Wnt3a的培养液中培养6d,分别在空白对照组,50μmol/L DAPT、0.1%DMSO、50μmol/L DAPT+25ng/mL Wnt3a培养液中继续培养7d,共培养14d。在所有培养过程中均加入10μmol/L Brdu。培养结束后Brdu,Myocin7a及细胞核进行免疫荧光染色。结果 实验第一部分：在25ng/mL Wnt3a组中可见β-catenin在支持细胞胞浆中有阳性表达,β-catenin阳性细胞数与其他各组比较差异具有统计学意义(P<0.01)。实验第二部分：DMSO组中可见Myosin7a染色阳性细胞,对照组、Wnt3a+DAPT组及DATP组中可见大量Brdu阳性细胞而未见Myosin7a染色阳性细胞,DMSO组中Myosin7a阳性细胞数与其他3组相比较,组间差异具有显著性(P<0.01)。结论 顺序联合应用Wnt3a和DMSO可通过激活经典WNT信号途径,促使椭圆囊内细胞向毛细胞样细胞转分化。     

碱性成纤维细胞生长因子对豚鼠椭圆囊毛细胞再生的影响

目的 利用豚鼠椭圆囊培养的方法观察碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对毛细胞再生的影响。方法 应用新霉素破坏豚鼠离体椭圆囊毛细胞,实验组培养液含有bFGF,对照组培养液则不含有bFGF。培养18d后两组均行扫描电镜检查,并行新生毛细胞计数,比较两组新生毛细胞数目的差异,观察bFGF对椭圆囊毛细胞再生的影响。结果 实验组新生毛细胞数目明显多于对照组(P<0.05)。结论bFGF对椭圆囊毛细胞的再生有促进作用。     

光学相差显微镜下前庭囊斑两型毛细胞的形态学鉴别

目的:探讨电生理实验中,光学相差显微镜下较为可靠的前庭两型毛细胞辨别标准.方法:对于豚鼠椭圆囊和球囊分离所得的前庭毛细胞,分别对细胞胞体的长、宽以及表皮板纤毛的长度进行测量,并对长宽比和纤毛胞长比进行分析.结果:豚鼠椭圆囊和球囊的两型前庭毛细胞相比,细胞的宽度和纤毛的长度比较差异无统计学意义,Ⅰ型毛细胞的长度明显较长,长宽比较大,纤毛胞长比较小.结论:在既往以是否有明显的颈部来区分两型前庭毛细胞的基础上,结合细胞的长宽比和纤毛胞长比进一步进行辨别,有助于更准确地区分两型毛细胞.     

实验动物前庭感觉毛细胞的定量观察

目的 测量常用实验动物内耳前庭感觉区的实际面积和量化分析前庭各个感觉区的毛细胞总数或密度。方法 ①制作CBA/CaJ小鼠、裸鼠、SD大鼠、豚鼠、南美栗鼠、新西兰白兔和非洲黑长尾猴的球囊斑铺片和椭圆囊斑铺片及壶腹嵴铺片,所有铺片样品来自每种受试动物的6个颞骨,在放大100倍的光学显微镜下拍摄2个囊斑铺片的整体照片;②应用Image J软件的图像测量程序,测量了上述7种常用实验动物球囊斑和椭圆囊斑的实际面积;③用网格将球囊斑铺片和椭圆囊斑铺片照片上的前庭感觉区划分为一个个方块区域。在放大400倍的光学显微镜下准确计数每个方格内的毛细胞数量,然后将每个方格的毛细胞计数结果相加以获得每种受试动物球囊斑和椭圆囊斑上的毛细胞总数;④应用前庭小视野定量观察技术计算出前庭各个感觉区小视野范围内的毛细胞密度。结果 ①从小鼠、裸鼠、大鼠、豚鼠、南美栗鼠、白兔到猴的球囊斑面积依次为(0.193±0.009)、(0.216±0.008)、(0.323±0.010)、(0.528±0.035)、(0.687±0.065)、(1.237±0.075)、(1.371±0.032)mm²;椭圆囊斑的面积依次为(0.193±0.020)、(0.208±0.013)、(0.321±0.011)、(0.526±0.034)、(0.795±0.017)、(1.224±0.082)、(1.388±0.048)mm²;②从小鼠、裸鼠、大鼠、豚鼠、南美栗鼠、白兔到猴的球囊斑毛细胞的总数依次为(2476.3±64.4)、(2389.8±47.8)、(3135.3±191.6)、(4882.2±208.7)、(6128.5±242.9)、(10572.2±464.4)、(10992.7±397.4)个;椭圆囊斑毛细胞的总数依次为(2491.4±54.8)、(2368.0±46.1)、(3218.8±82.9)、(4925.3±271.1)、(7794.0±386.1)、(11347.4±435.7)、(11114.5±410.6)个;③从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的球囊斑微纹区和周边区的毛细胞密度(毛细胞数量/0.007mm²)依次为101.0±5.79(微纹区)/120.8±4.15(周边区),95.5±3.91(微纹区)/109.2±5.26(周边区),78.4±6.54(微纹区)/94.8±4.38(周边区),60.0±4.74(微纹区)/84.6±2.61(周边区),57.2±3.83(微纹区)/80.0±3.54(周边区),53.8±4.21(微纹区)/68.0±4.18(周边区)。从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的椭圆囊斑微纹区和周边区的毛细胞密度(毛细胞数量/0.007mm²)依次为103.8±5.02(微纹区)/119.2±3.70(周边区),91.2±2.49(微纹区)/106.4±4.16(周边区),74.1±3.54(微纹区)/90.8±3.56(周边区),60.4±4.98(微纹区)/81.6±2.07(周边区),57.8±1.92(微纹区)/77.8±3.70(周边区),54.0±2.74(微纹区)/66.4±2.51(周边区)。从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的壶腹嵴毛细胞密度(毛细胞数量/0.007mm²)依次为112.4±6.38,105.5±3.51,95.2±3.42,84.0±7.16,78.2±2.86,70.8±2.39。可见由于体型较小动物毛细胞的细胞体比体型较大动物毛细胞的细胞体小,因而体型较小动物的前庭毛细胞密度高于体型较大动物的前庭毛细胞密度。另外,每种实验动物球囊斑和椭圆囊斑微纹区的毛细胞密度相似,周边区的毛细胞密度也大致相同,但是同种实验动物囊斑微纹区的毛细胞密度却低于周边区的毛细胞密度。此外,壶腹嵴毛细胞的密度与球囊斑和椭圆囊斑周边区的毛细胞密度几乎相同。鉴于某些损害因素往往具有选择性破坏囊斑微纹区毛细胞的表现,因此囊斑微纹区的毛细胞密度应该与囊斑周边区的毛细胞密度区分开来进行统计,必要时甚至需要把Ⅰ型毛细胞和Ⅱ型毛细胞也区分开来分别予以病理学改变的定量评估。结论 本研究采用的前庭测量方法和获得的前庭各个感觉区的测量数据和毛细胞总数及毛细胞密度,为前庭病理学研究的定量分析提供了有益的参考经验和必要的参考数据。     

内淋巴囊上皮细胞透明质酸合成酶mRNA的表达

目的 研究内淋巴囊上皮细胞透明质酸合成酶（ｈｙａｌｕｒｏｎａｎｓｙｎｔｈａｓｅ）ｍＲＮＡ的表达及其意义。方法 在查阅核酸序列数据库基础上,采用Ｍｏｔｉｆ程序分析各物种（大鼠,小鼠,爪蟾,人类）透明质酸合成酶的保守序列,应用原位杂交技术检测透明质酸合成酶ｍＲＮＡ在内淋巴囊上皮细胞的表达。结果 内淋巴囊近侧端,中间部均有部分上皮细胞胞浆显示透明质酸合成酶ｍＲＮＡ的阳性表达。结论 在分子生物学水平证实了     

Immunoelectron microscopic and immunofluorescent localization of cytoskeletal and muscle-like contractile proteins in inner ear sensory hair cells

The distribution of actin, alpha-actinin, fimbrin, tropomyosin and tubulin in the apical region of inner and outer hair cells was studied by immunofluorescent localization of antibodies to these proteins. The macromolecular distribution of actin and alpha-actinin was studied using post-embedding immunoelectron microscopic techniques. Actin is present in the stereocilia and cuticular plate of both inner and outer hair cells. Antibodies to actin were localized with fluorescence and colloidal gold. Colloidal gold particles were distributed uniformly over the stereocilia, stereocilia rootlets and cuticular plate. Fimbrin is present in the stereocilia and the cuticular plate. Immunofluorescent label was more intense over the cuticular plate of outer hair cells than over the cuticular plate of inner hair cells. Alpha-actinin is present in the cuticular plate only. At the ultrastructural level, antibodies to alpha-actinin were labeled throughout the cuticular plate, with larger accumulations of colloidal gold over the electron dense bodies in the cuticular plate, as well as over the electron dense region at the junctional complex. There was no label over the electron dense portion of the stereocilia rootlets. Tropomyosin is observed in the area of the stereocilia rootlets by immunofluorescent techniques, but like fimbrin, the antigenic sites of tropomyosin did not withstand processing for ultrastructural localization. Tubulin is not present in the apical region of inner or outer hair cells, although its presence could be documented in the hair cell body and in the supporting cells.     

Growth of a fish ear: 1. Quantitative analysis of hair cell and ganglion cell proliferation

Proliferation (or addition) of inner ear sensory hair cells continues for a long time postembryonically in cartilaginous and bony fishes, and in amphibians. In contrast, proliferation only occurs during embryonic development in birds and mammals. However, detailed quantitative data on hair cell addition are not available for bony fishes. In order to quantify the extent of proliferation, we determined the number of sensory hair cells on the saccular sensory epithelium in specimens of the cichlid fish Astronotus ocellatus (the oscar) ranging from 2.0 to 19.0 cm in standard length (0.9-343 g). Ganglion cells were counted using serial sections of the saccular branch of the eighth nerve in animals of the same size range. The saccular macula of a 2.0 cm long (0.9 g) Astronotus contains approximately 5500 sensory hair cells; fish from 16 to 19 cm long have over 170 000 hair cells. The increase in number of sensory cells and the increase in both length and weight of the animals studied were statistically correlated (r2 = 0.8). The relative densities of saccular sensory cells in different epithelial regions remained constant in animals from 2.0 to 17 cm; in larger animals the cell density decreased somewhat. Based upon very conservative estimates of the rate of growth of Astronotus, we calculate that an average of 167 hair cells/day are added during the time when the cell population of the saccule increases. Ganglion cell number also increased approximately 4.8 times in the range of fish studied. The smallest animals in our study had about 150 ganglion cells per saccular epithelium, while the largest fish had over 600 ganglion cells. We estimate that the average ratio of hair cells to afferent fibers increases from about 30:1 in the smallest fish to over 300:1 in the largest animals.     

Stapes anomaly and cochlear sensory cell changes

Summary This paper reports the pathological findings in the cochleae of a 66-year-old man examined under a scanning electron microscope (SEM). Columella-shaped stapes, which were the only anomalous changes in the middle ear cleft, were found in both ears. Beside this, giant hair formation and fusion of stereocilia in the inner sensory cells were also observed in the apical part of the lower basal turn. Since the patient had no history of ototoxic drug use or other ear disease episodes, it is suggested that these inner hair cell changes might have been caused by the conductive disorder accompanying the stapes anomaly. Giant hair formation was discussed from the inner ear findings of 14 other cases examined by a SEM.     

人鼻腔纤毛上皮细胞的浸没培养

目的：浸没培养法培养人鼻腔纤毛上皮细胞,为纤毛相关研究及经鼻药物安全性评价等研究提供可靠的细胞学模型。方法：采用低温酶消化法,浸没培养人鼻腔纤毛上皮细胞,倒置相差显微镜观察细胞生长状况,扫描电镜及免疫细胞化学方法观察细胞融合及纤毛分化状态,高速数字化显微视频成像系统检测纤毛摆动频率。结果：①相差显微镜下,纤毛细胞数量逐渐增加,至7～10d达到高峰后逐渐减少,纤毛细胞存活时间维持在14～21d;②细胞培养第7天,扫描电镜可见鼻腔上皮细胞表面覆盖纤毛或微绒毛,杯状细胞及无纤毛柱状上皮细胞相间排列;③细胞培养第7天,Ⅳ型β-微管蛋白、闭合小环蛋白-1免疫荧光结果显示细胞融合及纤毛分化良好,纤毛细胞比例可达20％～30％;④培养第7、14、21天纤毛摆动基础频率分别为（10．73±2．15）Hz、（9．92±1．97）Hz、（10．30±2．11）Hz,无明显统计学差异;⑤外源性刺激剂100μmol／L ATP可明显增加纤毛摆动频率。结论：应用酶消化法浸没培养人鼻腔纤毛上皮细胞,细胞融合及纤毛分化状态良好,纤毛摆动活跃,对外源性刺激反应灵敏,是应用于纤毛相关研究及经鼻药物安全性评价等研究的一个较为理想的细胞模型。     

人鼻息肉黏膜上皮细胞3种原代体外培养方案的比较

目的:比较人鼻息肉黏膜上皮细胞3种原代体外培养方法的效果,探讨适宜的体外培养方案。方法:对人鼻息肉黏膜上皮细胞分别以酶消化分离法、酶消化分离细胞ABC法和组织块培养法进行原代体外培养,观察细胞形态,掌握其生物学特性,并对3种方案的培养成功率和细胞生长曲线进行比较。结果:酶消化分离细胞ABC法体外培养成功率为87.5%,优于酶消化分离法(78.13%)和组织块培养法(84.38%),但三者间差异无统计学意义(P>0.05)。酶消化分离法和酶消化分离细胞ABC法的细胞生长曲线无明显差异,均高于组织块培养法。结论:酶消化分离细胞ABC法细胞增殖迅速,无其他细胞混杂,可建立稳定、可靠的人鼻黏膜上皮细胞原代培养模型,可为鼻息肉的相关研究提供良好的细胞系。     

Ultrastructural correlates of selective outer hair cell destruction following kanamycin intoxication in the chinchilla

Kanamycin ototoxicity, combined with behavioral audiometry to evaluate threshold shifts, was used to destroy outer hair cells (OHCs) in the basal cochlea of the chincilla while leaving the inner hair cell (IHC) population largely intact. After survival times of four weeks to one year, transmission electron microscopy was employed to determine the condition of surviving hair cells and neural elements. Throughout the region of OHC loss, IHCs and their innervation were normal in appearance if their adjacent supporting cells were undamaged. When IHC supporting cells, specifically the inner pillar cells, were damaged or absent, damage to IHCs was commonly obsereved. Such supporting cell-related damage included extrusion of the cuticular plate from the surface of the reticular lamina, encapsulation and/or fusion of stereocilla, and gross distortion of hair cell shape. When the outer supporting cells of the organ of Corti were undamaged following OHC loss, outer spiral fibers were found to have survived in near-normal numbers in the region from 0.5–1.0 mm basalmost surviving OHC, but suffered progressive attrition toward the basal end of the cochlea. It is concluded that kanamycin-induced OHC loss can occur without concommitant IHC damage or outer spinal fiber loss.     

人鼻黏膜上皮细胞的原代培养及传代

目的探讨人鼻黏膜上皮细胞的原代培养及传代方法。方法取鼻内镜下行鼻中隔手术时切除的正常下鼻甲黏膜。以DMEM/F12培养基及Keratinocyte SFM（K SFM）培基先后培养及传代,同时培养鼻咽上皮细胞NP69作参照。于倒置显微镜下进行细胞形态学观察,以台盼蓝染色观察细胞活力,免疫细胞化学进行上皮细胞鉴定。结果鼻黏膜上皮细胞呈鹅卵石样生长,传代后形态一致性提高,细胞活力达88.1%,纯度达92.97%,免疫组织化学证实为上皮细胞。结论此方法培养的鼻黏膜上皮细胞活力及纯度高,并可进行一定程度的传代,是获得人鼻黏膜上皮细胞的一种有效方法。     

Outer hair cell loss and supporting cell expansion following chronic gentamicin treatment

A sequence of changes in the organ of Corti associated with the destruction of outer hair cells (OHCs) and their replacement by supporting cells following chronic gentamicin treatment has been examined using thin-sections and SEM. The progression of change of OHCs was matched by concomitant expansion of adjacent supporting cells. Hair cells ruptured in the lateral membrane. The apical fragment was retained in the reticular lamina and became surrounded basally by the expanded supporting cells. No large breaches at the surface of the organ of Corti were formed. Rather, it appeared that the tight junctions around the hair cell were maintained until junctions were established between newly adjacent supporting cells in the space once occupied by the hair cell body. Only then was the OHC apex disrupted and the debris released into the sub-tectorial space. Some features of the OHC degeneration process were reminiscent of the controlled, cellular self-destruction phenomenon of apoptosis. The results suggest the possibility that the processes of hair cell loss and replacement may be controlled enabling maintenance of permeability barriers during structural reorganisation.