干细胞在听觉损伤治疗中的运用前景

目的:总结近年来国内外有关干细胞在修复毛细胞丢失和损伤的研究近况,探讨干细胞替代疗法在听觉损伤方面的研究进展。资料来源:应用计算机检索Elsevier数据库1990-01/2005-05相关听觉损伤及干细胞修复的文献,检索词“stemcellsandhearingcellregeneration”。资料选择:对搜集的文献进行筛选。纳入标准:①毛细胞损伤与丢失。②听觉损伤与修复。③干细胞替代疗法治疗听觉损伤。排除标准:①干细胞的致瘤性。②综述文献、重复研究。资料提炼:共收集54篇关于听觉损伤修复文献,纳入17篇符合标准的文献,9篇文献对干细胞运用进行分析评价。资料综合:内耳细胞尤其是毛细胞及相关的神经元损伤丢失可导致听觉损伤和平衡觉失调。由于哺乳动物前庭和耳蜗毛细胞的再生修复能力非常有限,因此通常认为毛细胞的损伤丢失是导致感音神经性聋的主要成因,并且靠自身是无法再生与修复。细胞替代疗法在治疗耳聋性疾病方面具有广阔的运用前景,是近年来听觉研究领域十分关注的热点。移植治疗耳聋的最佳方法是利用耳源性干细胞。最近,研究者分别从刚出生大鼠的柯蒂氏器和成年小鼠的前庭椭圆囊分离出胚胎样多分化潜能的干细胞,内耳干细胞将是重塑毛细胞再生和恢复听觉功能的重要细胞源。结论:干细胞在治疗毛细胞丢失引发的听觉损伤方面具有重要价值,随着研究的深入细胞替代疗法必然呈现诱人的前景。     

干细胞治疗感音神经性聋的研究理论及其临床应用进展

背景:内耳毛细胞或螺旋神经元的丢失可引起感音神经性聋.内耳细胞死亡后,利用干细胞研究来恢复听力已成为近年来研究热点.目的:总结近年来干细胞向内耳细胞方向分化的体内外研究进展,对干细胞进行感音神经性聋后内耳细胞替代治疗的研究成果进行综述.方法:应用计算机检索Pubmed数据库(2000-01/2009-08),检索词为"inner ear,Stem cells";应用计算机检索CNKI数据库(2000-01/2009-08),检索词为"内耳、干细胞".结果与结论:计算机初检得到170篇文献,纳入与干细胞在感音神经性聋的细胞转化有密切关系的实验研究和综述,排除重复研究类文献,对符合条件的32篇文献进行汇总分析.不同类型干细胞具有向内耳细胞分化的能力,能够分化为神经源性细胞类型.干细胞移植到内耳结构中可以发生迁移、分化,并部分分化为损伤区域的细胞类型,为感音神经性聋听力恢复提供了一条治疗途径.如何使分化细胞发挥正常生理功能,移植到内耳后能否建立神经通路,是干细胞治疗感音神经性聋下一步研究方向.     

Biological therapy for the inner ear

Biological therapy for the inner ear has the potential to revolutionise the treatment of sensorineural hearing loss, the most common form of deafness. Progress in the molecular understanding of hearing and hearing loss, combined with advances in the fields of both gene and cellular therapy for the inner ear, is providing a robust foundation from which clinical translation is plausible. Potential areas of interest in gene therapy and its preclinical application to deafness are reviewed, and experimental progress that has occurred in cellular therapy for the inner ear is examined.     

Biological therapy for the inner ear

Biological therapy for the inner ear has the potential to revolutionise the treatment of sensorineural hearing loss, the most common form of deafness. Progress in the molecular understanding of hearing and hearing loss, combined with advances in the fields of both gene and cellular therapy for the inner ear, is providing a robust foundation from which clinical translation is plausible. Potential areas of interest in gene therapy and its preclinical application to deafness are reviewed, and experimental progress that has occurred in cellular therapy for the inner ear is examined.     

豚鼠脂肪间充质干细胞植入耳聋模型修复听力的作用

背景：脂肪间充质干细胞是否是治疗因毛细胞退化、缺失所造成的感音神经性聋的福音呢？目的：探讨豚鼠脂肪间充质干细胞经耳蜗鼓阶途径植入感音神经性耳聋动物模型后对听力的修复作用。方法：庆大霉素腹腔注射建立豚鼠感音神经性耳聋动物模型,耳蜗鼓阶途径植入豚鼠脂肪间充质干细胞,分别于植入后1,3周检测听性脑干反应,观察植入脂肪间充质干细胞后耳聋动物听力的变化;并追踪EDU标记的豚鼠脂肪间充质干细胞在耳蜗内的迁移及分布情况。结果与结论：在植入后1周及3周进行听性脑干反应检测,听力较移植前逐渐好转。植入细胞后1周,细胞大多分布在外淋巴液中,部分迁移至耳蜗柯蒂器贴附于基底膜上,植入细胞后3周,细胞不仅迁移并贴附在Corti器基底膜发挥作用,而且部分迁移到蜗神经,植入时间越长,存活细胞越少。结果表明豚鼠脂肪间充质干细胞通过耳蜗鼓阶途径微孔植入,可以定向迁移并存活最终达到提高听力的目的。     

Treatment of peripheral sensorineural hearing loss: gene therapy

Noise, chemicals and genetic defects are all common causes of irreversible hearing loss, which at present have no cure. Gene therapy may soon be utilized in both the protection and the treatment of these exogenous and endogenous sources of hearing loss. Gene therapy technology is rapidly developing and the inner ear is a particularly feasible model for gene therapy. This review outlines our current understanding of the mechanisms behind deafness and prospects for treatment, discusses the inner ear model in detail and reviews the efforts that have been made in inner ear gene therapy. Finally, the proposed next steps will be discussed. The viral mediated delivery of neurotrophins and antioxidants offers imminent promise in preventing and treating exogenous hearing loss and improving cochlear implant therapy.     

新生豚鼠神经干细胞的分离培养及鉴定和标记

背景:神经干细胞为内耳移植治疗感音神经性聋带来希望,而豚鼠是氨基甙类药物中毒性耳聋动物模型的首选动物,且经过几十年研究已建立了成熟的动物模型。如何从豚鼠海马中提取神经干细胞并标记,是内耳干细胞移植所要解决的问题之一。目的:从新生豚鼠海马中分离培养神经干细胞,为神经干细胞移植治疗感音神经性耳聋的实验研究创造条件。设计:单一样本观察。单位:郑州大学微生物与免疫教研室。材料:实验于2005-03/06在郑州大学微生物与免疫教研室完成。新生24h内豚鼠(由郑州大学实验动物中心提供)50～80g/只,雌雄均有。方法:分离新生豚鼠海马组织,用含碱性成纤维生长因子和表皮生长因子无血清培养技术培养神经干细胞,免疫组织化学技术检测其巢蛋白的表达。采用荧光染料DAPI标记神经干细胞。主要观察指标:①观察神经干细胞体外生长情况。②免疫荧光检测鉴定神经干细胞巢蛋白表达。③荧光标记干细胞并检测标记率。结果:①从新生豚鼠海马组织分离出的细胞中可获得呈集落样生长的神经干细胞团。②神经干细胞能表达巢蛋白。③荧光染料DAPI的标记效率可达93.4%,细胞传代8次后荧光亮度仍无明显衰减。结论:从新生豚鼠海马分离的细胞具有明显的增殖能力,荧光染料的标记可获得较高的标记效率,可作为神经干细胞移植治疗感音神经性耳聋实验研究的供体细胞。     

Sudden deafness and cytomegaly virus infection (author's transl)]

A 49-year old patient complained of sudden deafness in his right ear. With the exception of a rising titre level of the complement-fixing antibodies there were no positive findings for the cytomegaly virus. However, the eight-fold rise in the titre level after a latent period made it appear likely that the sudden deafness was caused by a cytomegaly virus infection. This assumption was also supported by the lack of vestibular symptoms, which, according to literature, is quite frequent in virus diseases of the inner ear. The usual therapy led to an improvement in the patient's hearing, which was also accompanied by a drop in the titre level of the antibodies. No prognosis can be made, since similar observations have not yet been recorded.     

Acupuncture effect on neurosensory deafness.

Bilateral neurosensory deafness offers a unique opportunity to study the effects of acupuncture objectively. By treating only one ear, and using audiometric evaluation techniques instead of subjective evaluations of hearing, an analysis of any achieved improvement seemed feasible. Only patients with well documented bilateral nerve deafness were accepted into the program. A baseline audiogram with speech discrimination and puretone air and bone conduction thresholds was obtained prior to treatment. Thirty-eight patients underwent ten consecutive acupuncture treatments on one ear only, with the other ear serving as a control. After the course of acupuncture, the audiometrics were repeated by the same audiologist. There was no significant increase in hearing after acupuncture therapy.     

A “Tric” to tighten cell-cell junctions in the cochlea for hearing

Tricellulin is a tricellular tight junction–associated membrane protein that controls movement of solutes at these specialized cell intersections. Mutations in the gene encoding tricellulin, TRIC, lead to nonsyndromic deafness. In this issue of the JCI, Nayak et al. created a gene-targeted knockin mouse in order to mimic the pathology of a human TRIC mutation. Deafness appears to be caused either by an increase in the K⁺ ion concentration around the basolateral surfaces of the outer hair cells or, alternatively, by an increase in small molecules such as ATP around the hair bundle, leading to cellular dysfunction and degeneration. Furthermore, the mice have features suggestive of syndromic hearing loss, which may have implications for care and treatment of patients harboring TRIC mutations. Millions worldwide suffer from a debilitating hearing loss (1). In many regions of the world in which health care conditions and public health are less developed or in populations with high rates of consanguinity, the number of people affected is extremely high. Understanding the mechanisms leading to hearing loss may help widen the current scope of therapeutic options, which are currently restricted to hearing aids and cochlear implants. The causes of hearing loss are manifold, including both genetic and environmental factors. Hereditary hearing loss is caused by mutations in a wide variety of genes that encode the proteins associated with the transduction of sound waves from the external ear to the middle and inner ear and finally to the brain. To date, hereditary hearing loss has been linked to over 60 genes (2). The cochlea, located in the inner ear, is responsible for the conversion of sound to a neural electric excitatory signal (3). Two distinct fluids, the endolymph and perilymph, are contained within the cochlea. While the perilymph has an ionic composition similar to that of the general extracellular fluid, the endolymph is characterized by a high K⁺ concentration and endocochlear potential (EP), which are essential for hearing (4). An epithelial cellular sheet covering the cochlea creates a barrier that allows these fluids to maintain their composition.     

Current status and prospects of gene therapy for the inner ear

Inner ear diseases are common and often result in hearing disability. Sensorineural hearing loss is the main cause of hearing disability. So far, no effective treatment is available although some patients may benefit from a hearing aid equipped with a hearing amplifier or from cochlear implantation. Inner ear gene therapy has become an emerging field of study for the treatment of hearing disability. Numerous new discoveries and tremendous advances have been made in inner ear gene therapy including gene vectors, routes of administration, and therapeutic genes and targets. Gene therapy may become a treatment option for inner ear diseases in the near future. In this review, we summarize the current state of inner ear gene therapy including gene vectors, delivery routes, and therapeutic genes and targets by examining and analyzing publications on inner ear gene therapy from the literature and patent documents, and identify promising patents, novel techniques, and vital research projects. We also discuss the progress and prospects of inner ear gene therapy, the advances and shortcomings, with possible solutions in this field of research.     

干细胞移植豚鼠耳蜗形态结构及听力的变化

背景:既往研究证明耳蜗微造孔注射病毒或非病毒载体行基因治疗对耳蜗结构及听功能的影响轻微.目的:观察骨髓间充质干细胞通过耳蜗微造孔术移植到正常豚鼠内耳后对耳蜗结构及听功能的影响.方法:正常豚鼠分3组,空白对照组未予任何干预;单纯耳蜗微造孔术组单纯行耳蜗微造孔术,不注射任何成分;骨髓间充质干细胞组鼓阶微造孔后注射经DAPI荧光标记的骨髓间充质干细胞.结果与结论:单纯耳蜗微造孔术组、骨髓间充质干细胞组经耳蜗微造孔术后7 d的听性脑干诱发电位阈值均较空白对照组提高,术后28 d恢复至正常水平;耳蜗石蜡切片苏木精-伊红染色显示实验动物耳蜗内结构无明显异常改变.骨髓间充质干细胞组耳蜗切片可见荧光信号多分布于耳蜗底周的外淋巴腔(鼓阶和前庭阶),无堆积堵塞管腔情况.结果表明经鼓阶开窗行骨髓间充质干细胞移植对耳蜗形态结构和听力的影响是轻微的,干细胞移植后能在耳蜗内迁移并存活.     

Sound preconditioning therapy inhibits ototoxic hearing loss in mice

Therapeutic drugs with ototoxic side effects cause significant hearing loss for thousands of patients annually. Two major classes of ototoxic drugs are cisplatin and the aminoglycoside antibiotics, both of which are toxic to mechanosensory hair cells, the receptor cells of the inner ear. A critical need exists for therapies that protect the inner ear without inhibiting the therapeutic efficacy of these drugs. The induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death and hearing loss. We hypothesized that exposure to sound that is titrated to stress the inner ear without causing permanent damage would induce HSPs in the cochlea and inhibit ototoxic drug–induced hearing loss. We developed a sound exposure protocol that induces HSPs without causing permanent hearing loss. We used this protocol in conjunction with a newly developed mouse model of cisplatin ototoxicity and found that preconditioning mouse inner ears with sound has a robust protective effect against cisplatin-induced hearing loss and hair cell death. Sound therapy also provided protection against aminoglycoside-induced hearing loss. These data indicate that sound preconditioning protects against both classes of ototoxic drugs, and they suggest that sound therapy holds promise for preventing hearing loss in patients receiving these drugs.     

Surgical management of congenital conductive deafness

Today's otologic surgeon has the opportunity to restore normal serviceable hearing in patients with congenital conductive deafness using tympanoplasty with ossicular chain reconstruction, stapedectomy, or fenestration of the horizontal semicircular canal. The particular surgical approach used is determined by the abnormalities found in the congenitally deformed ear. I report a series of 18 patients who had operation for congenital conductive deafness during a four-year period at the Farrior Clinic. The study results show the efficacy of surgical reconstruction in producing an improvement in hearing.     

IL-1β inhibition in autoimmune inner ear disease: can you hear me now?

Clinical vignette: A 51-year-old man with right-sided sudden hearing loss presents to the otology clinic. He has a 4-year history of episodic vertigo of several hours’ duration and fluctuating, progressive sensorineural hearing loss in his left ear. The vertigo attacks have not occurred for the last 18 months, and the left ear hearing is consistently poor. The patient’s right ear hearing has dropped in the last 36 hours. MRI imaging of brain and temporal bone are normal. A 2-week “burst and taper” of oral prednisone is administered with no effect. Over the next 3 months, serial audiograms show rapidly progressive loss of threshold and word recognition scores on the right side. A trial of high-dose prednisone (60 mg/d for 30 days) results in full recovery of the right ear hearing and substantial improvement in the left ear. As the prednisone dose is slowly tapered over several months, the hearing drops again.The patient above is likely suffering from autoimmune inner ear disease (AIED), a rare form of sensorineural hearing loss (SNHL). In 1979, McCabe provided the first clinical evidence that SNHL could have an autoimmune component (1), supporting the hypothesis put forth by Lehnhardt (2). Despite multiple confirmatory reports that have refined the clinical characterization of AIED, the pathogenesis of this very uncommon condition remains vague and controversial, in large part because there are no universally accepted diagnostic criteria or tests. A loose consensus has gradually arisen that AIED presents as an idiopathic, rapidly progressive, bilateral SNHL. AIED patients exhibit a loss of hearing that occurs over a period of weeks to months, too slow to be considered “sudden” SNHL and too fast to be classified as age-related or other genetically determined degenerative SNHL. Serial audiometry demonstrates progression, which is usually monotonic but may fluctuate. Typically the ears progress asynchronously, often years apart. In many cases, deafness in the first ear may be congenital or the result of some identifiable cause prior to rapid hearing loss in the second ear, raising the possibility of AIED. This rapidly progressive asynchronous hearing loss alone is considered sufficient by some to make an AIED diagnosis, whereas others require evidence of steroid responsiveness.     

The Influence of Chronic Undernutrition on Bone Growth in Children

Any surgical approach to deafness must limit its scope to conduction deafness. The established otologic surgical procedures were designed mainly to treat uncomplicated or complicated ear infections or to prevent acquired deafness that is due to infection.

The newer procedures are designed to reconstruct or restore middle ear structures and to improve hearing in children with conduction deafness.     

Effect of transgenic GDNF expression on gentamicin-induced cochlear and vestibular toxicity

Gentamicin administration often results in cochlear and/or vestibular hair cell loss and hearing and balance impairment. It has been demonstrated that adenovirus-mediated overexpression of glial cell line-derived neurotrophic factor (GDNF) can protect cochlear hair cells against ototoxic injury. In this study, we evaluated the protective effects of adenovirus-mediated overexpression of GDNF against gentamicin ototoxicity. An adenovirus vector expressing the human GDNF gene (Ad.GDNF) was administered into the scala vestibuli as a rescue agent at the same time as gentamicin, or as a protective agent, 7 days before gentamicin administration. Animals in the Rescue group displayed hearing thresholds that were significantly better than those measured in the Gentamicin or Ad.LacZ/Gentamicin groups. In the Protection group, Ad.GDNF afforded significant preservation of utricular hair cells. The data demonstrated protection of the inner ear structure, and rescue of the inner ear structure and function against ototoxic insults. These experiments suggest that inner ear gene therapy may be developed as a clinical tool for protecting the ear against environmentally induced insults.     

Atoh1 gene therapy in the cochlea for hair cell regeneration

Introduction: The sensory epithelium of the cochlea is a complex structure containing hair cells, supporting cells and auditory nerve endings, all of which degenerate after hearing loss in mammals. Biological approaches are being considered to preserve and restore the sensory epithelium after hearing loss. Of particular note is the ectopic expression of the Atoh1 gene, which has been shown to convert residual supporting cells into hair cells with restoration of function in some cases.

Areas covered: In this review, hair cell development, spontaneous regeneration and hair cell regeneration mediated by Atoh1 gene therapy in the cochlea are discussed.

Expert opinion: Gene therapy can be safely delivered locally to the inner ear and can be targeted to the sensory epithelium of the cochlea. Expression of the Atoh1 gene in supporting cells results in their transformation into cells with the appearance and function of immature hair cells but with the resulting loss of the original supporting cell. While the feasibility of Atoh1 gene therapy in the cochlea is largely dependent on the severity of the hearing loss, hearing restoration can be achieved in some situations. With further advances in Atoh1 gene therapy, hearing loss may not be as permanent as once thought.     

诱导多能干细胞的研究现状及展望

背景：同胚胎干细胞一样,诱导多能干细胞可以自我更新并可分化成各种类型的体细胞。诱导多能干细胞不仅在替代疗法上有重要价值,还可用于体外疾病模型的建立,以方便疾病机制的研究、药物的监测以及新治疗方法的检验。目的：综合分析诱导多能干细胞的方法。方法：应用计算机检索PubMed及中国期刊全文数据库2006年1月至2011年12月与诱导多能干细胞有关的文章,英文检索词为“induced pluripotent stem cells”;中文检索词为“诱导多能干细胞”,并限定文章语言种类为中文。共收集到330篇相关文献,排除内容陈旧或重复的文献,选用其中的31篇作为参考文献。结果与结论：文章从基因导入诱导多能干细胞、蛋白诱导多能干细胞、小分子诱导多能干细胞、诱导多能干细胞株间的特性和差异、提高诱导多能干细胞的转化率、诱导多能干细胞在动物疾病模型治疗中的应用等方面进行了阐述。研究发现具有适当匹配转录因子的体细胞是生成诱导多能干细胞的一个潜在实用的起始点。基因诱导多能干细胞还存在未被解决的问题,而蛋白诱导多潜能干细胞的方法不会影响细胞的遗传物质。将诱导多能干细胞诱导技术和转基因动物技术相结合,可进行定向变异和育种,提高动物的遗传本质,加快动物群体遗传变异程度。诱导多能干细胞不仅在替代疗法上有重要价值,还可用于体外疾病模型的建立,以方便疾病机制的研究、药物的监测以及新治疗方法的检验。     

分阶段添加细胞诱导因子诱导豚鼠脂肪间充质干细胞定向分化内耳毛细胞

背景：感音神经性耳聋主要是由内耳毛细胞的缺失或受损造成,用脂肪间充质干细胞来再生修复内耳毛细胞是治疗听力损失的有效方法。目的：探讨体外定向诱导豚鼠脂肪间充质干细胞向内耳毛细胞样细胞分化的可行性。方法：体外分离培养豚鼠脂肪间充质干细胞至第3代,流式细胞仪检测细胞免疫表型,分阶段加入表皮生长因子、碱性成纤维细胞生长因子、胰岛素样生长因子1、全反式维甲酸、脑源性神经营养因子、神经营养因子3,观察诱导分化的效果。结果与结论：豚鼠脂肪间充质干细胞体外培养呈梭形,漩涡状贴壁生长,第3代细胞表面标记CD29与CD44表达呈现阳性,CD34与CD45表达呈现阴性。应用细胞因子诱导后细胞早期nestin和GFAP表达阳性,继续诱导10 d后表达毛细胞特异性标记物MyosinⅦa和Math1,说明细胞因子可定向诱导豚鼠脂肪间充质干细胞向内耳毛细胞分化。