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.     

干细胞向耳蜗毛细胞及螺旋神经节细胞诱导分化研究

随着干细胞研究的深入,世界各国科学家已证明多种干细胞可向耳蜗毛细胞及螺旋神经节诱导分化,采用的方法也不尽相同.本文从细胞种类及诱导方法两方面就干细胞向耳蜗毛细胞及螺旋神经节细胞诱导分化的研究做一综述.     

哺乳动物内耳毛细胞损伤再生性修复的研究进展

耳聋是严重影响人类生活质量的顽疾之一，目前尚无根治的方法。各种疾病、噪声、药物等所致感音性耳聋。其实质均在于内耳毛细胞的变性和坏死。鱼类和两栖动物内耳毛细胞因噪声、外伤等因素损伤丢失后，在听觉和前庭器官能自发生成新的毛细胞，从结构和功能上修复受损的感觉上皮。Stone等研究听觉受损的鸟类发现。这些再生的毛细胞可能来源于邻近支持细胞的再生性增殖与分化。通常认为，哺乳动物出生后内耳毛细胞失去再生能力。由毛细胞丢失引发的耳聋是不可治愈的。但也有研究表明。由于外伤和药物作用导致听觉受损可引发哺乳动物内耳前庭器官恢复一定的再生能力嘲。这种再生毛细胞可能有以下几种来源：（1）受损或损伤区周围的支持细胞向毛细胞转化；（2）一些感觉上皮细胞的再分裂；（3）听觉上皮以外的某种干细胞（stemcells）激活，增殖并分化为毛细胞。上述因素中。一般认为支持细胞是新生毛细胞的前体细胞。支持细胞向新生毛细胞转化并替代坏死毛细胞是毛细胞损伤修复的关键因素。     

459细胞凋亡与内耳毛细胞的损伤方式

细胞凋亡对细胞的增殖，器官的发生和功能维持起着重要作用，近期研究发现，细胞凋亡也是内耳感觉上皮毛细胞的一种重要的损伤方式，并可能与感觉上皮细胞的增殖过程有关，本综述了内耳感觉上皮毛细胞损伤方式和毛细胞的凋亡，探讨内耳毛细胞的损伤方式与感觉上皮修复的关系。     

活性氧与内耳毛细胞凋亡相关的信号转导及基因调控

许多内耳疾病中均可检测到不同程度的毛细胞凋亡及相关的信号转导途径和基因的表达调控,而活性氧也在耳蜗组织损伤中发挥重要作用。本文对活性氧与内耳组织损伤、内耳细胞凋亡及其相关信号转导和基因调控和两者之间的关系等方面进行综述。     

药物耳毒性与内耳细胞凋亡

药物耳毒性是造成内耳前庭、耳蜗上皮细胞损伤的重要因素,表现为多种损伤方式.本文就药物耳毒性的机制和损伤方式作一综述,着重就药物耳毒性与细胞凋亡的关系进行探讨.     

体外诱导人脐带间充质干细胞向内耳毛细胞样细胞分化的研究

目的 探讨并建立一种体外定向诱导人脐带间充质干细胞(human umbilical cord mesenchymal stem cells ,hUC-MSCs)向内耳毛细胞样细胞分化的方法.方法 采用组织贴壁法培养获得hUC-MSCs,流式细胞仪鉴定其表面标记物;神经干细胞诱导阶段分为对照组(DFNB基础诱导培养基)、实验1组(DFNB基础诱导培养基+20 ng/ml EGF+20 ng/ml bFGF)、实验2组(DFNB基础诱导培养基+20 ng/ml EGF+20 ng/ml bFGF+琼脂糖包被),培养3~5天后采用免疫荧光法检测各组标记蛋白Nestin的表达;内耳毛细胞样细胞诱导分化阶段,分为对照组(DFNB基础诱导培养基+明胶包被)、诱导组[DFNB基础诱导培养基+20 ng/ml EGF+1 μM全反式维甲酸/全反式维生素A酸(all trans retinoic aid,ATRA)],培养4周后采用qRT-PCR和免疫荧光法检测各组毛细胞标记物Math1、MyosinⅦa、Brn3c的表达.结果 在神经干细胞诱导阶段,对照组、实验1组均无神经球结构,实验2组中hUC-MSCs分化3 d后细胞有明显神经球样结构,且Nestin阳性细胞数更高(P<0.05);而向内耳毛细胞样诱导分化4周后,诱导组毛细胞标记物Math1、MyosinⅦa、Brn3c的mRNA和阳性细胞数的表达水平明显升高(P<0.05).结论 体外诱导人脐带间充质干细胞先向神经干细胞方向分化再向内耳毛细胞分化方向诱导,可有效提高毛细胞标记物MyosinⅦa、Brn3c、Math1的表达水平,促进hUC-MSCs向类毛细胞样细胞分化,可能为将来感音神经性聋的内耳移植治疗提供更为适合的种子细胞.     

神经干细胞与乳鼠基底膜共培养后分化为毛细胞样细胞的研究

目的探讨体外培养和鉴定胚胎大鼠神经干细胞及诱导其分化为毛细胞样细胞。方法从SD系胚鼠大脑分离神经干细胞，在无血清培养基中培养。取乳鼠基底膜与神经干细胞一起培养，14—21天后通过免疫荧光和免疫组化法检测毛细胞标志物myosin VIIa和calretinin。结果培养的神经干细胞胞体透亮，折光性好，Nestin鉴定阳性。诱导分化后的细胞免疫荧光和免疫组化示myosin VIIa和calretinin阳性。结论无血清条件下能培养出活性很好的神经干细胞，乳鼠基底膜能引导其朝毛细胞方向分化。     

支持细胞在内耳发育及耳蜗毛细胞损伤后的作用

耳蜗毛细胞损伤后如何再生及恢复其听功能是现今耳科学研究的热点问题之一。目前国内外的研究热点为调控参与内耳毛细胞调控的基因、干细胞移植、生长因子诱导、内耳前体细胞向毛细胞分化等。对于螺旋器中支持细胞在内耳发育、耳蜗毛细胞损伤后保持听觉上皮完整以及毛细胞再生中的作用关注较少。本文就支持细胞在内耳发育、毛细胞损伤后的作用等研究做一综述。     

诱导性多能干细胞与内耳移植

感音神经性聋是耳科常见病,其发病机制是内耳毛细胞及螺旋神经节细胞的减少或缺失所致,目前对于这一疾病的治疗研究中细胞移植已成为热点,特别是诱导性多能干细胞的发现为感音神经性聋的治疗带来了新的希望。本文就诱导性多能干细胞与内耳移植进行综述。     

Regenerated Synapses Between Postnatal Hair Cells and Auditory Neurons

Regeneration of synaptic connections between hair cells and spiral ganglion neurons would be required to restore hearing after neural loss. Here we demonstrate by immunohistochemistry the appearance of afferent-like cochlear synapses in vitro after co-culture of de-afferented organ of Corti with spiral ganglion neurons from newborn mice. The glutamatergic synaptic complexes at the ribbon synapse of the inner hair cell contain markers for presynaptic ribbons and postsynaptic densities. We found postsynaptic density protein PSD-95 at the contacts between hair cells and spiral ganglion neurons in newly formed synapses in vitro. The postsynaptic proteins were directly facing the CtBP2-positive presynaptic ribbons of the hair cells. BDNF and NT-3 promoted afferent synaptogenesis in vitro. Direct juxtaposition of the postsynaptic densities with the components of the preexisting ribbon synapse indicated that growing fibers recognized components of the presynaptic sites. Initiation of cochlear synaptogenesis appeared to be influenced by glutamate release from the hair cell ribbons at the presynaptic site since the synaptic regeneration was impaired in glutamate vesicular transporter 3 mutant mice. These insights into cochlear synaptogenesis could be relevant to regenerative approaches for neural loss in the cochlea.     

Influence of Supporting Cells on Neuronal Degeneration After Hair Cell Loss

In sensorineural hearing loss, hair cell loss is often followed by loss of cochlear nerve fibers, which can continue for years after the insult. The degree and time course of neuronal loss varies, but the reasons for this variation are unclear. The present study addresses this issue with a quantitative analysis of hair cell, supporting cell, and neuronal survival in animals with long-term survival of up to 5.5 years from two types of drug-induced hair cell loss: aminoglycoside antibiotics and platinum-containing chemotherapeutics. To complement the analysis of the effects of organ of Corti damage on neuronal survival, cases of primary neuronal degeneration, via auditory nerve section, are also assessed. Analysis shows that (1) long-term neuronal survival is enhanced when supporting cells in the inner hair cell (IHC) area remain intact; (2) after hair cell loss, the time course of neuronal loss is slower in the apex than in the base; (3) primary loss of cochlear nerve fibers does not lead to secondary degeneration of sensory cells or supporting cells in the organ of Corti; and (4) after auditory nerve section, there can be a massive reinnervation of the IHC region, especially in the apex. Results are consistent with the idea that supporting cells participate in the regulation of neuronal survival and neuronal sprouting in the organ of Corti.     

微吸管单类细胞收集法对哺乳动物内耳细胞转录组的研究

目的本文介绍使用微吸管技术分类收集高纯度的内耳毛细胞的方法。方法将成年小鼠内耳基底膜与Corti’s器一起分离,使用胶原酶消化后获得游离单个细胞,采用微吸管对内耳毛细胞、支持细胞进行分类收集、提取总RNA,并进行单类细胞成分的转录组分析。结果分离后的成年小鼠内耳毛细胞、支持细胞形态独特、易于辨认,可以进行有效采集,所获得的内耳单类细胞样本纯度高、质量好。结论采用微吸管方法对哺乳动物内耳毛细胞进行单类细胞收集是一种可行、有效的实验方法,此方法的应用使得对成年哺乳动物内耳单类细胞转录组的研究成为可能。     

Reciprocal Innervation of Outer Hair Cells in a Human Infant

Reciprocal synapses are characterized by the presence of both afferent and efferent types of synaptic specializations between two cells. They have been described at the neural poles of outer hair cells (OHCs) in humans with advanced age and two monkey species. Our objective was to study the innervation of the OHCs and determine if reciprocal synapses were present in a young (8-month-old infant) human subject. We studied the synaptic and cytoplasmic morphology of 162 nerve terminals innervating 29 OHCs using serial section transmission electron microscopy. Seventy-six percent of all OHCs were innervated by terminals with reciprocal synapses. This prevalence increased from the first toward the third row (p < 0.001), and 100% of OHCs in the third row demonstrated at least one reciprocal synapse. The prevalence of terminals with reciprocal synapses was higher in the human infant than in older human subjects and was very similar to what has been reported for the chimpanzee. Reciprocal synapses occur in sufficient numbers to be physiologically significant in primates. The nerve terminals were found to segregate into two groups on the basis of their cytoplasmic morphological characteristics: (1) vesicle-rich/neurofilament-poor (VR/NP) and (2) vesicle-poor/neurofilament-rich (VP/NR). All afferent and reciprocal terminals were of the VP/NR variety. The majority of the efferent terminals originated from VR/NP nerve fibers (classical olivocochlear morphology), but 23.5% of the efferent terminals were VP/NR. The hypothesis that peripheral processes of type II spiral ganglion cells form classical afferent, reciprocal, and a number of purely presynaptic terminals on OHCs is discussed. The presence of different types of synaptic specializations on OHCs formed by nerve fibers of the same type (VP/NR) suggests the existence of reciprocal neuronal circuits between OHCs sharing the dendritic arborization of a type II spiral ganglion cell.     

Serial Section Reconstruction of the Neural Poles of Hair Cells in the Human Organ Of Corti. I. Inner Hair Cells

Study of the anatomy of the cochlea, and in particular the morphology of synaptic relationships between hair cells and cochlear neurons, is essential for elucidation of the mechanisms of transduction of mechanical acoustic signals into electrical neural events. Because considerable gaps remain in our understanding of the microscopic anatomy of these synapses, particularly in the human, a reconstruction of the neural pole of inner hair cells of the human organ of Corti was performed. The data are based on 526 serial sections from the basal turn (10 mm region) and 356 serial sections from the middle turn (26 mm region). This provided complete data on 3 and partial data on 5 inner hair cells. Afferent terminals on inner hair cells were variable in size, ranging 1 to 20 µm in diameter. Branching of large fibers to produce multiple terminals innervating from 1 to 3 inner hair cells was common. Each inner hair cell received approximately 6 to 8 different nerve terminals. In addition, each terminal possessed a variable number of synaptic contacts. Junctional membrane specialization consisted of synapses, desmosomes, coated vesicles and arrays of microtubules and membrane cisternae. Specialization at synapses consisted of asymmetrical membrane thickening. At inner hair cells the postsynaptic membrane was thicker than the presynaptic membrane. Eighty-three percent of synapses had presynaptic bodies. Vesiculated efferent terminals synapsed on afferent fibers at the base of inner hair cells, but never directly on the inner hair cell. These anatomical data demonstrate distinct differences between the human and animal inner ear, which are important in the interpretation of neurophysiological data in animals and the formulation of hypotheses that involve assumptions crossing species.     

Differences Between the Negatively Activating Potassium Conductances of Mammalian Cochlear and Vestibular Hair Cells

Cochlear and type I vestibular hair cells of mammals express negatively activating potassium (K⁺) conductances, called g _K,n and g _K,L respectively, which are important in setting the hair cells resting potentials and input conductances. It has been suggested that the channels underlying both conductances include KCNQ4 subunits from the KCNQ family of K⁺ channels. In whole-cell recordings from rat hair cells, we found substantial differences between g _K,n and g _K,L in voltage dependence, kinetics, ionic permeability, and stability during whole-cell recording. Relative to g _K,L, g _K,n had a significantly broader and more negative voltage range of activation and activated with less delay and faster principal time constants over the negative part of the activation range. Deactivation of g _K,n had an unusual sigmoidal time course, while g _K,L deactivated with a double-exponential decay. g _K,L, but not g _K,n, had appreciable permeability to Cs⁺. Unlike g _K,L, g _K,ns properties did not change (wash out) during the replacement of cytoplasmic solution with pipette solution during ruptured-patch recordings. These differences in the functional expression of g _K,n and g _K,L channels suggest that there are substantial differences in their molecular structure as well.     

Supporting Cell Division Is Not Required for Regeneration of Auditory Hair Cells After Ototoxic Injury In Vitro

In chickens, nonsensory supporting cells divide and regenerate auditory hair cells after injury. Anatomical evidence suggests that supporting cells can also transdifferentiate into hair cells without dividing. In this study, we characterized an organ culture model to study auditory hair cell regeneration, and we used these cultures to test if direct transdifferentiation alone can lead to significant hair cell regeneration. Control cultures (organs from posthatch chickens maintained without streptomycin) showed complete hair cell loss in the proximal (high-frequency) region by 5 days. In contrast, a 2-day treatment with streptomycin induced loss of hair cells from all regions by 3 days. Hair cell regeneration proceeded in culture, with the time course of supporting cell division and hair cell differentiation generally resembling in vivo patterns. The degree of supporting cell division depended upon the presence of streptomycin, the epithelial region, the type of culture media, and serum concentration. On average, 87% of the regenerated hair cells lacked the cell division marker BrdU despite its continuous presence, suggesting that most hair cells were regenerated via direct transdifferentiation. Addition of the DNA polymerase inhibitor aphidicolin to culture media prevented supporting cell division, but numerous hair cells were regenerated nonetheless. These hair cells showed signs of functional maturation, including stereociliary bundles and rapid uptake of FM1-43. These observations demonstrate that direct transdifferentiation is a significant mechanism of hair cell regeneration in the chicken auditory after streptomycin damage in vitro.     

斑马鱼毛细胞损伤及再生研究进展

内耳毛细胞的损伤是引起感音神经性聋的常见原因,而寻找刺激毛细胞再生的可能条件,是治疗感音神经性聋的重要方向;斑马鱼作为新兴模式生物,其胚胎及早期幼鱼通体透明,侧线毛细胞位于体表,以及生殖周期短、繁殖能力强且基因与人类基因约87%相似。鉴于斑马鱼以上生物学特性,耳科学研究者亦采用斑马鱼模型探讨毛细胞损伤及再生机制。本文拟对斑马鱼毛细胞损伤及再生的相关研究进展进行综述。     

Preliminary Characterization of Voltage-Activated Whole-Cell Currents in Developing Human Vestibular Hair Cells and Calyx Afferent Terminals

We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11–14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15–18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G _K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a “collapsing” tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15–18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.     

脑源性神经营养因子基因修饰骨髓基质细胞对体外培养小鼠螺旋神经元的影响

目的探讨人脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因工程细胞的建立及其对体外培养的小鼠螺旋神经元生长活性的影响。方法参照分子克隆技术成功构建BDNF真核表达载体———pcD-NA3.1(-)-BDNF,体外证实BDNF基因转染猿猴病毒40肝脏转化细胞(COS7)后正常表达,将骨髓基质细胞分离培养,体外转染骨髓基质细胞建立BDNF基因工程细胞,观察该基因工程细胞对体外培养小鼠螺旋神经元的生长活性的影响。结果成功构建BDNF基因真核表达载体,并且建立了BDNF基因工程细胞;在体外BDNF基因工程细胞能促进螺旋神经元的生长,并保护螺旋神经元免受氧化损伤。结论建立的BDNF基因修饰的骨髓基质细胞,对体外螺旋神经元生长、生存起着重要的保护作用,为进一步研究基因修饰的骨髓基质细胞内耳移植奠定了重要的基础。