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。     

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。     

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.     

大鼠椭圆囊感觉上皮细胞长期培养体系的建立及毛细胞标记物表达的意义

目的：建立椭圆囊感觉上皮细胞（USEC）长期培养体系，为内耳毛细胞再生研究提供稳定的活性细胞。方法：取1d龄wistar大鼠，在显微镜下取出椭圆囊上皮，嗜热菌蛋白酶处理，获得具活性的纯椭圆囊感觉上皮，胰蛋白酶＋胶原酶消化，培养传代，传25代。取第25代USEC在倒置显微镜、透射电镜下对USEC的生长特征、形态结构进行观察；免疫细胞化学检测细胞角蛋白18、波形蛋白、Brn3．a及Calretinin的表达。RT—PCR检测毛细胞的特征性标记物AchRa9、MyosinVIIamRNA的表达。结果：USEC细胞培养达6个月传25代，第25代USEC均呈扁平、多角形、核大而圆的上皮细胞形态，细胞之间连接紧密，形成单层时均呈“铺路石样”外观，可见dome结构。该细胞表达角蛋白18和不表达波形蛋白，微绒毛丰富，细胞间连接紧密，提示其上皮起源。第25代USEC表达毛细胞的特征性标志物Brn3．a、Calretinin及AchRa9、Myosin Ⅶa mRNA，表明长期培养USEC仍具有毛细胞的前体细胞的特性。结论：本实验成功建立了USEC长期培养体系。长期培养的USEC性状未发生明显改变，表达上皮细胞及毛细胞的特征性标志物，具有毛细胞前体细胞的特征。这为毛细胞再生的机制及内耳分子、基因研究提供了稳定的细胞来源。     

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。     

豚鼠前庭椭圆囊器官在庆大霉素损伤后离体培养的细胞增殖

目的 在离体状态下对豚鼠前庭椭圆囊器官进行培养,观察庆大霉素损伤毛细胞后细胞的增殖情况。方法采用白色健康豚鼠36只,体重300-450克,随机分为4组:(1)正常对照组;(2)正常加BrdU(5-溴基脱氧尿核苷BrdU)组;(3)庆大霉素组;(4)庆大霉素加BrdU组。应用体外组织培养、5—溴基脱氧尿核苷(BrdU,3μg/ml)掺入及免疫组织化学染色和Epon树酯包埋半薄切片等方法,在不同时间(2、3、5、15天)光镜观察庆大霉素(2.0mM)损伤后毛细胞及支持细胞增殖情况。结果 用庆大霉素48小时后,可见到椭圆囊感觉上皮中的毛细胞溶解破坏。加入BrdU继续培养,BrdU免疫组织化学染色显示了第5和15天被BrdU标记的阳性细胞核,阳性细胞核多位于椭圆囊感觉上皮中基底层,从细胞的形态和分布位置上看为支持细胞;感觉上皮的表层,也可观察到BrdU反应阳性的上皮细胞,细胞核较圆,形态和位置类似毛细胞。结论 提示损伤后的椭圆囊感觉上皮支持细胞可以进行细胞的有丝分裂,支持细胞可能是修复毛细胞的前体细胞。     

二甲基亚砜对在体耳蜗毛细胞的毒性作用

目的研究二甲基亚砜(DMSO)对在体耳蜗毛细胞的毒性作用。方法取清洁级健康、ABR阈值正常SD大鼠32只,雌雄不限,体重100-120g。随机分成4组,人工外淋巴液对照组(即0%组)8只;0.1%DMSO溶液组8只;1%DMSO溶液组8只;5%DMSO溶液组8只。所有动物均取右耳作为实验耳。通过耳蜗鼓阶打孔显微注射向每个实验耳注入不同浓度DMSO溶液4ul。术前1天和术后7天分别进行ABR(click和toneburst)检测。激光共聚焦显微镜观察DMSO溶液导入7天后的毛细胞变化。结果人工外淋巴液对照组click、4kHz、8kHz、16kHz处阈移平均值均<5dBSPL,仅于32kHz处有约13dBSPL的阈移,形态方面未见明显损伤;0.1%浓度组在click、4kHz、8kHz处阈移平均值均<5dBSPL,而32kHz处阈移约25dBSPL,与人工外淋巴液对照组比较提示有统计学意义,激光共聚焦显微镜观察底回时偶见少数内毛细胞胀大;1%浓度即可引起OHC大量丢失,造成相应纤毛表皮板缺如,且以底回最重,各频率ABR阈移均>15dBSPL,32kHz处阈移>30dBSPL;当浓度增加到5%时,不仅损伤耳蜗底回的外毛细胞,也导致内毛细胞的丢失,所造成的听力损失在32kHz处较1%组严重。结论 DMSO对毛细胞的损伤存在剂量依赖性,损伤程度自耳蜗底回向顶回逐渐减轻。     

ATP对离体耳蜗外毛细胞内游离钙浓度的影响

为探讨三磷酸腺苷（ＡＴＰ）对耳蜗外毛细胞内游离钙（Ｃａ２＋）浓度的影响，利用荧光染色法，以５７０型粘附式细胞仪对ＡＴＰ引起的豚鼠耳蜗外毛细胞Ｃａ２＋浓度的变化进行动态观察。加入ＡＴＰ使Ｃａ２＋浓度明显升高，升高后迅速下降，高峰处无延迟，再次加入ＡＴＰ，出现类似反应。说明ＡＴＰ是以一种神经递质的作用影响外毛细胞的生理活动     

温度变化对豚鼠离体前庭毛细胞内钙离子浓度的影响

目的 为探讨温度改变诱发前庭眼震的机理。方法 酶孵育后机械分离成功豚鼠前庭毛细胞（ＶＨＣ）３４个，Ｆｌｕｏ－３荧光染色后，改变其外环境的温度，用激光扫描共聚焦显微镜（ＬＳＣＭ）记录静息状态下及温度改变时ＶＨＣ内钙离子浓度（［Ｃａ＾２＋］ｉ）的变化。结果 将分离的ＶＨＣ分为两型，ＬＳＣＭ下钙离子分布图像与相差显微镜下所见细胞外形相似，其钙离子分布在核区略高。１８／２２的Ｉ型与７／１２的Ⅱ型ＶＨＣ其细     

豚鼠椭圆囊斑静纤毛间的相互连接形式

目的：探讨豚鼠椭圆囊斑毛细胞静纤毛束间的相互连接形式及其生理意义。方法：用扫描电镜和透射电镜观察豚鼠椭圆囊斑毛细胞静纤毛束间相互连接。结果：豚鼠椭圆囊斑毛细胞静纤毛束间存在３种相互连接形式：①顶部连接：自短纤毛顶至长纤毛的上行连接;②侧连接：同行静纤毛间的云雾状连接;③行间连接：邻近行间的连接。结论：３种连接的存在可将纤毛束连为一体。推测当纤毛偏转时,所有静纤毛可能形成一个整体依次移动,牵拉或放松     

Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity

Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.     

β-肌动蛋白在雏鸡毛细胞再生修复过程中的变化

目的探讨雏鸡基底乳头（basilar     

庆大霉素致豚鼠前庭毛细胞破坏后再生及功能恢复

为探讨哺乳动物前庭毛细胞破坏后能否再生及前庭功能能否恢复，以豚鼠为研究对象。实验组动物每日给予庆大霉素（１５０ｍｇ／ｋｇ）皮下注射，连续１０天，对照组动物给予等量生理盐水。于停药后的第２、４、１２和２４周分别处死动物，扫描电镜观察发现，停药２周椭圆囊和壶腹嵴毛细胞破坏最明显的区域可见到不成熟的毛细胞纤毛丛，至停药２４周后继续存在。与对照组相比，停药１２周毛细胞密度（毛细胞数／１００μｍ基底膜长度）下降明显（Ｐ＜０．０１），停药后２４周时毛细胞密度较１２周明显上升（Ｐ＜０．０５），但仍未达到正常水平（Ｐ＜０．０５）。另取豚鼠按上述方法进行庆大霉素注射，于注射前和停药后４、１２和２４周进行冰水诱发眼震电图检查，可见停药４～１２周平均慢相眼震速度（Ｖｍｅａｎ）明显下降（Ｐ＜０．０１），停药２４周后Ｖｍｅａｎ有明显恢复（Ｐ＜０．０５），但仍未达到正常水平（Ｐ＜０．０１）。提示：豚鼠周围前庭功能破坏后可以部分恢复，前庭毛细胞破坏后可以修复，其功能的恢复可能源于毛细胞再生。     

Hair cell regeneration in senescent quail

Hair cell regeneration was studied following exposure to an intense pure tone stimulus in young adult and senescent Coturnix quail. Three, 3-month old and four, 3-year old quail were continuously exposed to a 1500 Hz pure tone at 115 dB SPL for 12 h. Four quail were not noise exposed and were used as age-matched controls. Control and experimental birds received injections of [³H]thymidine daily for 10 days after noise exposure. Ten days after noise exposure birds were killed and their cochleae embedded, sectioned serially and processed through standard methods of autoradiography.

Hair cell counts showed a discreet area of hair cell loss for both age groups in the proximal half of the papilla. Incorporation of [³H]thymidine was clearly seen over the nuclei of hair cells and support cells in the region of hair cell loss in both age groups. Incorporation of [³H]thymidine was also seen over the nuclei of hair cells and support cells in a very small area in two of the non-exposed control birds.

These results demonstrate that the potential for hair cell regeneration is maintained throughout life in Coturnix quail. Further, they suggest that there may be some very low level of hair cell production in the normal adult quail ear which is activated in the absence of massive trauma.     

Streptomycin ototoxicity and hair cell regeneration in the adult pigeon utricle

OBJECTIVE: The purpose of this study was to develop a technique to investigate the regeneration of utricular hair cells in the adult pigeon (Columba livia) following complete hair cell loss through administration of streptomycin. STUDY DESIGN: Experimental animal study. METHODS: Animals were divided into four groups. Group 1 received 10 to 15 days of systemic streptomycin injections. Animals in Groups 2 and 3 received a single direct placement of a 1-, 2-, 4-, or 8-mg streptomycin dose into the perilymphatic space. Animals in Groups 1 and 2 were analyzed within 1 week from injection to investigate hair cell destruction, whereas Group 3 was investigated at later dates to study hair cell recovery. Group 4 animals received a control injection of saline into the perilymphatic space. Damage and recovery were quantified by counting hair cells in isolated utricles using scanning electron microscopy. RESULTS: Although systemic injections failed to reliably achieve complete utricular hair cell destruction, a single direct placement of a 2-, 4-, or 8-mg streptomycin dose caused complete destruction within the first week. Incomplete hair cell loss was observed with the 1-mg dose. Over the long term, regeneration of the hair cells was seen with the 2-mg dose but not the 8-mg dose. Control injections of saline into the perilymphatic space caused no measurable hair cell loss. CONCLUSIONS: Direct placement of streptomycin into the perilymph is an effective, reliable method for complete destruction of utricular hair cells while preserving the regenerative potential of the neuroepithelium.     

人脂肪源性间充质干细胞向内耳毛细胞定向诱导分化的实验研究

目的 验证在体外将人脂肪源性间充质干细胞(human adipose-derived mesenchymalstem cells,hAD-MSC)定向分化为内耳毛细胞的可行性.方法 用特定的培养体系配合多种细胞因子定向诱导hAD-MSC向神经干/祖细胞样细胞分化,而后进一步将诱导后细胞与发育期鸡胚听泡细胞在体外进行共培养,以促使其向内耳毛细胞分化,通过免疫组化等方法对分化不同阶段的细胞特异性指标进行鉴定.结果 hAD-MSC诱导后呈现神经干/祖细胞样的形态并表达其特异性标志,与发育期鸡胚听泡细胞共培养后表达内耳毛细胞特异性标志.结论 hAD-MSC在体外可定向诱导分化为具有内耳毛细胞特异性标志的毛细胞样细胞.     

体外诱导大鼠骨髓间充质干细胞向内耳毛细胞样细胞分化的研究

目的 探讨体外定向诱导大鼠骨髓间充质干细胞向耳蜗毛细胞样细胞分化的可行性.方法 分离培养大鼠骨髓间充质干细胞,并加以鉴定.分离出生1～3 d的乳鼠耳蜗Corti器,与骨髓间充质干细胞在体外共培养14 d.通过反转录聚合酶链反应(RT-PCR)和免疫细胞化学染色对分化细胞的特异性分子指标(myosinⅦa、math1及calretinin)进行鉴定.结果 免疫细胞化学染色显示诱导分化后的细胞myosinⅦa、math1和calretinin表达阳性,RT-PCR提示分化后的细胞可表达毛细胞的标志物myosinⅦa和math1.结论 体外培养的骨髓间充质干细胞可诱导分化为具有毛细胞分子标志物的毛细胞样细胞.     

荧光下豚鼠听觉离体毛细胞的凋亡改变

目的：探讨离体耳蜗毛细胞的凋亡改变。方法：采用胶原酶制备离体耳蜗毛细胞、0．01％吖啶橙进行荧光染色,荧光显微镜观察凋亡改变。结果：凋亡毛细胞的主要形态特征包括：胞核皱缩;荧光显微镜下凋亡毛细胞呈红色或红黄色荧光,毛细胞形态完整,无胞膜破裂或胞质溢出。结论：离体耳蜗毛细胞的凋亡改变使细胞呈红色或红黄色荧光,胞膜完整。