Inner ear hair cell regeneration in a mammal: Identification of a triggering factor

Recent observations have shown that mammals possess a limited capacity for regeneration of inner ear sensory epithelia. It is clear, however, that a mitogenic growth factor will be necessary to up-regulate this capacity before clinical application becomes feasible. This study used in vitro cultures of adult mouse vestibular organs for assessing the mitogenic effect of transforming growth factor alpha (TGF-α). Sixty-one utricles and cristae were maintained in culture for 7 to 8 days. Neomycin was used to damage the hair cells. Autoradiography permitted identification of any cell which had undergone mitosis during the culture period. The proliferative response was compared in organs exposed to TGF-alpha and those maintained in the basic culture medium only. The results demonstrated that TGF-alpha significantly increased cell proliferation in the sensory epithelia and also in the marginal zones surrounding them. This finding provides a scientific basis for the concept that inner ear hair cell damage in humans may someday be reversible pharmacologically.     

ErbB Expression: The Mouse Inner Ear and Maturation of the Mitogenic Response to Heregulin

In humans, hair cell loss often leads to hearing and balance impairments. Hair cell replacement is vigorous and spontaneous in avians and nonmammalian vertebrates. In mammals, in contrast, it occurs at a very low rate, or not at all, presumably because of a very low level of supporting cell proliferation following injury. Heregulin (HRG), a member of the epidermal growth factor (EGF) family of growth factors, is reported to be a potent mitogen for neonatal rat vestibular sensory epithelium, but its effects in adults are unknown. We report here that HRG- stimulates cell proliferation in organotypic cultures of neonatal, but not adult, mouse utricular sensory epithelia. Our findings support the idea that the proliferative capabilities of the adult mammalian vestibular sensory epithelia differ significantly from that seen in neonatal animals. Immunohistochemistry reveals that HRG-binding receptors (erbBs 2–4) and erbB1 are widely expressed in vestibular and auditory sensory epithelia in neonatal and adult mouse inner ear. The distribution of erbBs in the neonatal and adult mouse ear is consistent with the EGF receptor/ligand family regulating diverse cellular processes in the inner ear, including cell proliferation and differentiation. Present address (Mette Kirkegaard): Department of Zoophysiology, Bld. 131, Universitetsparken, University of Aarhus, Denmark     

Epidermal growth factor receptor expression on oral mucosa dysplastic epithelia and squamous cell carcinomas

Summary The expression of the receptor for epidermal growth factor (EGF) has been determined on oral squamous cell carcinomas. Immunoreactive receptor was localized using a monoclonal anti-EGF-receptor antibody which reacts with sequences in the external domain of the receptor. Frozen sections were studied from 40 patients with squamous cell carcinomas. In 16 sections from the patients with the squamous cell carcinomas, normal differentiated oral mucosa was included and in 7 of these the patients had received preoperative radiotherapy. Sections from 6 other patients with squamous cell carcinoma contained dysplastic epithelia. EGF-receptor-positive cells were present in the basal cell layer on normal differentiated oral mucosa. In sections from patients receiving preoperative radiotherapy the EGF-receptor-positive cells were also found in the spinous cells. In dysplastic epithelia nearly all cells stained for the receptor. The distribution and staining intensity of the EGF receptor varied in the oral squamous cell carcinomas, 36 were positive. The staining pattern in the carcinomas obtained from patients receiving preoperative radiotherapy was not altered qualitatively. Nearly all poorly differentiated cells were stained, but when the tumor was moderately to well differentiated a reduction in the extent of staining in certain areas was seen, paralleling the findings observed in the differentiated upper layers of the normal oral mucosa. This was most pronounced for the epithelial pearls, where the EGF-receptor-positive cells were localized to the undifferentiated cells in the periphery. The results of the present investigation confirm the presence of the EGF receptor on undifferentiated cells, with the extent of the staining reaction on oral squamous cell carcinomas varying inversely with cellular differentiation.     

The role of middle ear effusions and epidermal growth factor in cholesteatoma formation in the gerbilline temporal bone

To study the process of aural cholesteatoma formation, we used gerbilline temporal bones to examine histologically the early stages of spontaneous cholesteatomas associated with experimentally induced otitis media with effusion (OME) following electric cauterizations of the eustachian tube. Epidermal growth factor (EGF) was then localized immunohistochemically in the pars flaccida of normal ears and the forming spontaneous cholesteatomas. Findings in the ears with the early spontaneous cholesteatomas were effusion inside the pars flaccida and hypertrophy and hyperkeratosis of the pars flaccida. Findings in the ears with experimental OME involved an effusion in the whole middle ear cavity as well as hypertrophy and hyperkeratosis in both the pars flaccida and pars tensa. The incidence of ear drum changes was higher in the experimental OME group than in control animals without cauterization. EGF was localized in the mucous layer of normal drums, the mucous layer and lamina propria of drums with hypertrophy alone, and all lalers in drums with hypertrophy and hyperkeratosis. EGF was especially positive in the cytoplasms of transformed cuboidal cells. These findings suggest that EGF within the transformed mucous layer may play an important role as a biochemical factor in developing cholesteatomas.     

Actin-associated proteins and fibronectin in the fetal human inner ear

The distribution of alpha-actinin, vinculin, alpha-spectrin, beta-spectrin and fibronectin was analyzed in 14- to 21-week-old fetal human inner ears using immunofluorescence microscopy. Staining for alpha-actinin was fairly evenly distributed at the epithelial surfaces of all five vestibular organs, whereas in the cochlea it was mainly at the surface of the receding greater epithelial ridge and in some foci apically at the lesser epithelial ridge. Fluorescence for vinculin was observed mainly at the surface of vestibular organs, but was lacking in the LER. Intense fluorescence for alpha-spectrin was found at the apical surface of individual cells of the cristae and maculae. Antibodies against beta-spectrin mainly stained the endothelial cells of blood vessels, but faint staining of the epithelial cell surfaces of the vestibular organs was also detected. The fluorescence pattern of the actin-associated proteins is indicative of structural differences between cochlear and vestibular hair cells. Fibronectin was identified only between mesenchymal cells and its functional importance in the mature inner ear epithelia can be discounted.     

表皮生长因子及地塞米松对爆震性聋治疗作用的实验研究

为研究爆震后豚鼠耳蜗毛细胞表皮生长因子受体（epidermalgrowthfactorreceptor，EGFR）的表达，以及表皮生长因子（epidermalgrowthfactor，EGF）及地塞米松（dexamethasone，DXM）的治疗效果，采用免疫组织化学ABC技术和听性脑干反应（ABR）检测。结果发现正常豚鼠耳蜗内、外毛细胞有散在的表皮生长因子受体（EGFR）表达，阳性反应位于听毛，呈节段性分布。爆震后24小时EGFR阳性反应分布于内毛细胞胞浆。爆震后72小时内毛细胞听毛和外毛细胞皮板呈阳性反应。震后1周内毛细胞阳性反应明显增多，部分外毛细胞听毛呈阳性反应。EGFR阳性反应至2周有所减少。震后1个月内、外毛细胞听毛均呈EGFR阳性反应。EGF及DXM对爆震性聋均有一定的治疗作用，二者合用可使豚鼠听力明显恢复。结果提示，毛细胞损伤修复可能与EGF有关。     

Immunohistochemical detection of epidermal growth factor receptor in laryngeal squamous cell carcinomas.

Laryngeal squamous cell carcinomas from 15 consecutive preoperatively irradiated patients were investigated for the expression of epidermal growth factor receptor (EGF receptor). The study was performed on frozen sections by means of the 5-layer APAAP technique employing an antibody recognizing the extracellular part of the EGF receptor. In sections from 9 of the patients with laryngeal squamous cell carcinoma, normal differentiated epithelia were included. Sections from 6 of these patients, in addition, contained dysplastic epithelia. Expression of EGF receptor-like material was demonstrated in the basal cell layer of normally differentiated laryngeal epithelial and in dysplastic epithelia. Fourteen of the squamous cell carcinomas proved EGF receptor positive. Nearly all cells in the poorly differentiated carcinomas showed positive staining with the antibodies. In moderately to well differentiated carcinomas a reduction in the extent of staining was seen in certain areas. Especially for the epithelial pearls, the staining reaction was localized to the undifferentiated cells in the periphery. This finding corresponds to the staining pattern observed in the basal cell layers of normal epithelial. The present investigation confirms the expression of EGF receptor-like material in normal laryngeal epithelial, dysplastic epithelial and squamous cell carcinoma. The staining pattern was similar to that observed in oral squamous cell carcinomas, predominantly varying inversely with cellular differentiation.     

Comparative study of epidermal growth factor and observation only on human subacute tympanic membrane perforation

Objective

To compare the effects of epidermal growth factor (EGF) and observation only on human subacute tympanic membrane perforation (TMP).

Expression of epidermal growth factor receptor, transforming growth factor-alpha and Ki-67 in relationship to malignant transformation of pleomorphic adenoma

CONCLUSION: Quantitative assessment is more sensitive as a measure of cellular protein content as compared with standard optical density measurements. The data support the hypothesis that increased epidermal growth factor receptor (EGFR) and transforming growth factor (TGF)-alpha expression is associated with early events in malignant transformation of pleomorphic adenoma (PA). OBJECTIVE: In the present study, we attempted to identify EGFR and TGF-alpha expression and Ki-67 index in carcinoma ex-pleomorphic adenoma (Ca ex-PA) and PA. We also compared the presence of EGFR and TGF-alpha and Ki-67 index with clinical data. MATERIALS AND METHODS: The tissues were stained with monoclonal antibodies to EGFR, TGF-alpha and Ki-67. The results were analysed using quantitative immunohistochemical analysis. We also analysed the association of patients' prognosis with clinical parameters and the histological classification of the carcinomatous component. RESULTS: As regards the association of patients' prognosis with EGFR staining and Ki-67 index, a significant increase was observed in patients who died or had residual disease compared with patients who were alive without disease. In the immunohistochemical analysis of EGFR and TGF-alpha and Ki67 index, a significant increase was observed in Ca ex-PA, especially with adenocarcinoma, compared with PA and sialadenitis.     

表皮生长因子及其受体在中耳慢性鼓膜穿孔病变中的作用

OBJECTIVE: To evaluate the possible roles of epidermal growth factor(EGF) and its receptor (EGFR) on the chronic tympanic membrane perforations. METHODS: A phosphate buffer saline of EGFR was administered to a Gelfoam pledget placed over chronic tympanic membrane perforations in guinea pigs. The EGFR of 10 specimens from the acquired middle ear cholesteatoma of the adjacent skin around perforation was examined by immunohistochemical SP method and computer image analysis. Results Complete closure of the tympanic membrane perforations was observed in 82.6% of EGF-treated ears, but only 33.3% in the controls(P < 0. 01). No case was led to middle ear cholesteatoma in the experiment group (0/23). The positive expression in the adjacent skin around perforation was (39.3 -/+ 7.4)%; and the normal external ear skin was (25.4 +/- 3.7)%; There were distinctly significant differences between the adjacent skin around perforation and the normal external ear skin (P < 0. 01). CONCLUSIONS: EGF is effective on closing chronic tympanic membrane perforations in the guinea pigs. Present data suggests that EGF-treated may induce the occurrence of middle ear cholesteatoma.     

Role of sialoglycan structures for the function of the epidermal growth factor receptor and the in vitro proliferation of head and neck cancer

Squamous cell carcinomas of the head and neck have been found to show a high expression of the receptor for epidermal growth factor (EGF). This overexpression of the receptor has been associated with malignant transformation of cells, although there is still debate as to what extent this receptor takes part in the proliferation of malignant cells and which function it fulfills. The factors which determine receptor-ligand interaction are also not clearly defined. That the extracellular domain of the EGF receptor carries carbohydrate or sialoglycan structures might be important for function of the receptor. Since tumor specific enzymes can possibly alter such structures, it was the aim of our study to investigate the role of these structures on the EGF receptor during the proliferation of head and neck carcinomas. We used the human laryngeal squamous carcinoma cell line HLaC 79 and altered, for the first time, specific glycan structures with sialidase α-2,3 and α-2,6, causing desialylation. Changes were also produced by endo-β-galactosidase and sialyltransferase. Findings were monitored by labeling with bromo-deoxyuridine. To determine receptor affinity, ¹²⁵I-labeled EGF was employed. Results showed that both cell proliferation and receptor affinity depended on the level of sialylation of the receptor carbohydrate side chains. Desialylation led to a statistically significant reduction of tumor cell proliferation to 65 ± 33% (P < 0.01), while receptor affinity decreased to 70 ± 26% (P < 0.01).The importance of EGF receptor for the proliferation of malignant cells seems to depend on the level of sialylation of glycan structures on receptor protein. A release of enzymes by tumor cells may then produce auto-control of tumor proliferation on its own. Received: 5 November 1997 / Accepted: 21 April 1998     

表皮生长因子受体及血管内皮生长因子在喉乳头状瘤中的表达

目的探讨表皮生长因子受体（EGFR）和血管内皮生长因子（VEGF）在喉乳头状瘤（IJP）中的表达及意义。方法采用免疫组化二步法检测10例成人型喉乳头状瘤（ALP）、19例幼年型喉乳头状瘤（JLP）石蜡标本中EGFR、VEGF的表达与分布;并以10例声带息肉作为对照组。结果EGFR和VEGF在ALP、JLP组上皮层的表达水平明显高于对照组（P〈0．05）。EGFR在ALP、JLP表皮组织全层均有强阳性表达,VEGF呈现以基底层、棘层细胞显著表达,到颗粒层表达逐渐减弱的模式。VEGF在ALP、JLP和对照组间质的血管内皮细胞、炎症细胞、成纤维细胞中也有表达,但3组问VEGF的表达元显著统计学差异（P〉0．05）。JLP组上皮中VEGF的表达评分结果（7．133±0．061）比ALP组（6．934±0．041）高,两组比较有统计学意义（P〈0．05）。结论VEGF、EGFR在LP组织中的过度表达可能在LP的上皮细胞过度增生和血管大量形成中发挥重要作用,JLP比ALP具有更强的增殖活性。     

表皮生长因子在急性鼻窦炎黏膜修复过程中的作用及机制

目的观察表皮生长因子（Epidermal Growth Factor,EGF）对实验动物急性鼻窦炎鼻窦黏膜的愈合作用,以及其受体在鼻窦黏膜愈合过程中的表达变化。方法在鼻窦炎动物模型建立后将兔麻醉,打开上颌窦前壁,在内镜下于上颌窦底做一黏膜缺损区。左侧上颌窦为对照组,每天用生理盐水处理窦腔;右侧上颌窦为实验组,每天应用含EGF的生理盐水进行窦腔处理。并取未经任何处理的兔子作为正常组。在实验开始后1、2、3、4周,各随机处死6只动物,比较观察两侧上颌窦黏膜创面愈合情况,以及表皮生长因子受体表达情况。结果实验组EGF可有效促进鼻窦黏膜创面愈合,创面上皮化明显高于对照组,并且局部创缘表皮生长因子受体（EGFR）表达显著高于对照组。结论EGF可通过刺激局部组织EGFR的表达从而促进鼻窦黏膜上皮化。     

Pretreatment factors affecting traumatic tympanic membrane regeneration therapy using epidermal growth factor

Objective

The use of epidermal growth factor (EGF) to achieve closure of human traumatic tympanic membrane perforations (TMPs) was recently reported. However, pretreatment factors affecting healing outcomes have seldom been discussed. This study was performed to evaluate pretreatment factors contributing to the success or failure of TMP healing using EGF.

表皮生长因子受体信号通路在人鼻腔上皮RPMI-2650细胞中的作用

目的 通过对表皮生长因子受体(epidermal growth factor receptor,EGFR)信号通路不同部位的干预,观察体外培养的人鼻腔上皮细胞RPMI-2650中EGFR和黏蛋白5AC(mucin 5AC,MUC5AC)的变化.方法 对人鼻腔上皮细胞RPMI-2650进行体外培养,记录生长曲线,并行扫描电镜观察细胞超微结构.当细胞大部分融合时,将细胞分为四组,A组:在原Eagle最小必需培养基(Eagle's minimum essential media,EMEM)培液中继续培养;B组:加入人重组表皮生长因子(epidermalgrowth factor,EGF)25 ng/ml;C组:加入AG1478(EGFR选择性抑制剂)10 μmol/L,30 min后加人人重组EGF 25 ng/ml;D组:加入PD98059(p44/42MAPK选择性抑制剂)30μmol/L,30 min后加入人重组EGF 25 ng/ml.以上四组细胞均继续培养24 h后,分别进行细胞免疫和Western blotting实验,观察EGFR和MUC5AC蛋白在各组细胞中的表达.结果 细胞在培养第3天开始融合,第5～7天大量融合.扫描电镜可见细胞呈圆形或椭圆形,表面覆有大量微绒毛,当细胞融合时可变为梭形或多角形生长.EGFR蛋白在B组细胞中强表达,A组和D组细胞中有较强表达,而在C组中仅有弱表达,A、B、D组与C组平均吸光度值(A值)相比差异均具有统计学意义(P值均<0.01).而MUC5AC蛋白在B组细胞中有强表达,A组细胞中有较强表达,在C组和D组中仅为弱表达,B组与C、D组间相比吸光度值差异均具有统计学意义(P值均<0.01),而C组与D组之间差异无统计学意义(P>0.05).结论 从细胞和蛋白水平上发现,EGFR信号通路在RPMI-2650细胞中发挥作用.对该通路不同部位进行干预,细胞中EGFR和MUC5AC蛋白随之发生相应改变.证实了EGFR信号通路在鼻腔上皮细胞RPMI-2650中对MUC的分泌具有调节作用.     

Northern印迹法检测喉鳞状细胞癌中表皮生长因子受体mRNA的表达

目的 :探讨喉部鳞状细胞癌的发病机制。方法 :采用Northern印迹法检测喉部鳞状细胞癌中表皮生长因子受体mRNA的表达。结果 :在喉部鳞状细胞癌中可见 5 80 0mRNA异常表达。结论 :喉部鳞状细胞癌的生物学行为与其 5 80 0mRNA介导异常表达密切相关。     

Expression of calretinin by fetal otocyst cells after transplantation into damaged rat utricle explants

Severe damage by acoustic overstimulation or ototoxins induces inner ear hair cell loss, resulting in permanent hearing loss and balance disorders because hair cell regeneration scarcely occurs in the inner ear sensory organs of mammals. In this study, to evaluate the possibilities of cell transplantation therapy for damaged inner ear sensory organs, dissociated cell cultures of fetal otocyst cells (FOCs) were established from embryonic day 12.5 (E12.5) rat inner ears, and transplanted into gentamicin-treated explants of vestibular sensory epithelia. Two weeks after transplantation, immunohistochemical analysis demonstrated that some of the grafted FOCs survived within the vestibular sensory epithelia and expressed epitopes of calretinin. one of the hair cell marker proteins. These findings indicate that FOCs have the potential to migrate into damaged vestibular epithelia and differentiate into hair cell immunophenotypes. Cell transplantation therapy may be available for functional regeneration in inner ear diseases.     

表皮生长因子与其受体在鼻息肉上皮中的表达

目的 探讨表皮生长因子(EGF)及其受体(EGFR)在鼻息肉上皮中的表达及其意义。方法 收集鼻息肉标本25例, 正常下鼻甲黏膜组织10例, 采用免疫组化及实时荧光定量PCR技术, 观察EGF与EGFR在鼻息肉和正常下鼻甲黏膜组织中的表达情况。结果 与正常下鼻甲黏膜组织相比, EGF及EGFR的mRNA水平在鼻息肉组织中的表达下降。在免疫组化染色中, EGF及EGFR主要表达在上皮的基底层细胞, 而且在鼻息肉中的表达下降。结论 EGF及EGFR在正常鼻黏膜组织上皮的发生与修复中有着重要的作用。EGF及EGFR在鼻息肉中蛋白水平与mRNA水平表达的下降表明在鼻息肉中上皮修复功能的下调或缺失可能导致或促进鼻息肉的发生。     

The effect of epidermal growth factor on the pseudo-healing of traumatic tympanic membrane perforations

IntroductionTraumatic tympanic membrane perforations tend to heal spontaneously. However, in this study, several perforations exhibited abnormal healing, where the morphology of healing tympanic membranes differed from that of non-perforated tympanic membranes. Pseudo-healing of the tympanic membrane was characterized by the accumulation of thickened tissue in the perforated area.ObjectiveThe purpose of this study was to evaluate the utility of epidermal growth factor in cases showing pseudo-healing of traumatic tympanic membrane perforations.MethodsA total of 26 traumatic tympanic membrane perforations showing pseudo-healing were included in this study. In all cases, tissue that accumulated in the perforated area was removed, which subsequently caused a new perforation to form. An epidermal growth factor solution was applied to the tympanic membrane once daily to keep the tympanic membrane moist. Closure rates and times were evaluated at 6 months.ResultsDuring the 6 months follow-up period, two patients were lost. Of the remaining 24 patients, the closure rate was 100% (24/24) and the closure time was 6.1 ± 2.3 days (range: 3–12 days). The morphology of the healed tympanic membrane was not significantly different from that of the remnant tympanic membrane.ConclusionsPseudo-healing of traumatic tympanic membrane perforations affects sound conduction. This can be associated with various symptoms, including tinnitus, aural fullness, and ear discomfort. The excision of excessive epithelial tissue and topical application of epidermal growth factor can correct the pseudo-healing of traumatic tympanic membrane perforations.