RESUMO
During development, cranial neural crest (NC) cells display a striking transition from collective to single-cell migration and undergo a mesenchymal-to-epithelial transformation to form a part of the middle ear epithelial cells (MEECs). While MEECs derived from NC are known to control homeostasis of the epithelium and repair from otitis media, paracrine action of keratinocyte growth factor (KGF) promotes the growth of MEECs and induces middle ear cholesteatoma (cholesteatoma). The animal model of cholesteatoma was previously established by transfecting a human KGF-expression vector. Herein, KGF-inducing cholesteatoma was studied in Wnt1-Cre/Floxed-enhanced green fluorescent protein (EGFP) mice that conditionally express EGFP in the NC lineages. The cytokeratin 14-positive NC lineage expanded into the middle ear and formed cholesteatoma. Moreover, the green fluorescent protein-positive NC lineages comprising the cholesteatoma tissue expressed p75, an NC marker, with high proliferative activity. Similarly, a large number of p75-positive cells were observed in human cholesteatoma tissues. Injections of the immunotoxin murine p75-saporin induced depletion of the p75-positive NC lineages, resulting in the reduction of cholesteatoma in vivo. The p75 knockout in the MEECs had low proliferative activity with or without KGF protein in vitro. Controlling p75 signaling may reduce the proliferation of NC lineages and may represent a new therapeutic target for cholesteatoma.
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Middle-ear cholesteatoma (cholesteatoma) is a chronic otitis media with an enhanced proliferation of epithelial cells. Negative pressure in the middle ear is thought to be important for the etiology of cholesteatoma. However, the mechanism of cholesteatoma formation remains unclear. Integrin-linked protein kinase (ILK), an important modulator of actin cytoskeletal dynamics, interacts with extracellular matrix and results in the up-regulation of mechanotransduction effector Yes-associated protein (YAP). The L1 cell adhesion molecule (L1CAM) has recently been reported as an activator of the mechanotransduction effectors related to cell proliferation and migration. In this study, we demonstrated a stretch assay for middle-ear cultured cells and performed immunohistochemistry using antibodies against Ilk, Yap, and L1cam. The tympanic membrane was also analyzed within a new middle-ear negative-pressure animal model and human cholesteatoma tissues, using immunohistochemistry with antibodies against ILK, YAP, Ki-67, and L1CAM. The expression of cytoplasmic ILK and nuclear shift of YAP increased in the thickened epithelium of the tympanic membrane under a negative-pressure load and the cholesteatoma. The expression of L1CAM was detected in the stromal cells, which enhanced epithelial cell proliferation depending on ILK signaling events. In conclusion, we demonstrated the possibility that the stromal L1CAM and epithelial ILK-YAP signaling played an important role in epithelial growth under mechanotransduction in cholesteatoma formation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/fisiologia , Colesteatoma da Orelha Média/metabolismo , Células Epiteliais/metabolismo , Mecanotransdução Celular/fisiologia , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Colesteatoma da Orelha Média/patologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Camundongos , Membrana Timpânica/metabolismo , Membrana Timpânica/patologia , Proteínas de Sinalização YAPRESUMO
Promyelocytic leukaemia (PML) is a tumor suppressor protein covalently conjugated with SUMO family proteins, leading to the formation of PML nuclear bodies (NBs). PML-NBs provide a platform for efficient posttranslational modification of targets and protein-protein interaction, contributing to the adjustment of gene expression and chromatin integrity. Although PML SUMOylation is thought to play important roles in diverse cellular functions, the control mechanisms of adequate modification levels have remained unsolved. Here, we report that Cullin-related protein CACUL1/CAC1 (CACUL1) inhibits PML posttranslational modification. CACUL1 interacts with PML and suppresses PML SUMOylation, leading to the regulation of PML-NB size in the nucleus. We also found that Ubc9, a SUMO-conjugating enzyme, binds to CACUL1 and antagonizes the interaction between CACUL1 and PML. Furthermore, CACUL1 attenuates p53 transcriptional activity. These data suggest that CACUL1 is a novel regulator that negatively controls p53 activity through the regulation of PML SUMOylation.
Assuntos
Proteínas Culina/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Neoplasias/metabolismo , Mapas de Interação de Proteínas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Perioperative assessment and care, such as enhanced recovery after surgery (ERAS), is very important for improving the clinical outcomes of patients who have undergone surgery. However, professional assessments and care cannot be achieved through the actions of only 1 surgical department. We established a perioperative management center(PERIO) comprised of surgeons, dedicated nurses, anesthesiologists, dentists, physiotherapists, pharmacists, and nutritionists to perform intensive cross-sectoral perioperative management. In this manuscript, we investigated the impact of PERIO on the clinical outcomes of 127 elderly patients who underwent thoracic surgery for the resection of non-small cell lung cancer (NSCLC). We categorized these 127 patients into 3 groups:â those treated before the introduction of PERIO (between January 2006 to August 2008), â¡ those treated during the early phase after PERIO introduction (September 2008 to December 2011), and ⢠those treated during the late phase after PERIO introduction( January 2012 to December 2014). Radical operations were performed significantly more frequently after PERIO introduction than before PERIO introduction, while the postoperative complication rates were similar among the 3 groups. The duration of postoperative hospitalization was reduced after the introduction of PERIO, and the hospital surplus increased after the introduction of PERIO. In conclusion, PERIO may play an important role in improving the clinical outcomes of thoracic surgery, especially for elderly patients with NSCLC.
Assuntos
Período Perioperatório , Procedimentos Cirúrgicos Torácicos , Idoso de 80 Anos ou mais , Feminino , Humanos , Neoplasias Pulmonares/cirurgia , Masculino , Equipe de Assistência ao Paciente , Complicações Pós-OperatóriasRESUMO
Histone acetylation is involved in the regulation of chromatin structure and gene function. We reported previously that histone H3 acetylation pattern is subject to dynamic changes and limited to certain stages of germ cell differentiation during murine spermatogenesis, suggesting a crucial role for acetylation in the process. In the present study, we investigated the effects of hyper- and hypo-acetylation on spermatogenesis. Changes in acetylation level were induced by either in vivo administration of sodium phenylbutyrate, a histone deacetylase inhibitor, or by knockdown of histone acetyltransferases using short hairpin RNA plasmids transfection. Administration of sodium phenylbutyrate induced accumulation of acetylated histone H3 at lysine 9 and lysine 18 in round spermatids, together with spermatid morphological abnormalities and induction of apoptosis through a Bax-related pathway. Knockdown of steroid receptor coactivator 1, a member of histone acetyltransferases, but not general control of amino acid synthesis 5 nor elongator protein 3 by in vivo electroporation of shRNA plasmids, reduced acetylated histone H3 at lysine 9 in round spermatids, and induced morphological abnormalities. We concluded that the proper regulation of histone H3 acetylation levels is important for spermatid differentiation and complex chromatin remodeling during spermiogenesis.
Assuntos
Histonas/metabolismo , Espermátides/patologia , Testículo/fisiopatologia , Acetilação/efeitos dos fármacos , Animais , Apoptose , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Tamanho do Órgão , Fenilbutiratos/farmacologia , Espermátides/efeitos dos fármacos , Espermátides/enzimologia , Espermátides/ultraestrutura , Espermatogênese/genética , Testículo/efeitos dos fármacos , Testículo/enzimologia , Testículo/ultraestruturaRESUMO
We reported previously that keratinocyte growth factor (KGF), a mesenchymal cell-derived paracrine growth factor, plays an important role in middle ear cholesteatoma formation, which is characterized by marked proliferation of epithelial cells. Here, we investigated whether KGF, the main factor that induces cholesteatoma, overexpression in vivo results in the formation of cholesteatoma. Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal (EAC) of rats once (short-term model) or five times on every fourth day (long-term model). Ears transfected with empty vector were used as controls. Successful transfection of plasmids into epithelial and stromal cells was confirmed by Flag immunohistochemistry. In the short-term model, the intensity of KGF protein was the strongest in hKGF transfected ear at day 4. KGF expression induced epithelial cell proliferation, reaching a peak level at day 4 and then decreased later, while in the long-term model, KGF expression in the EAC led to middle ear cholesteatoma formation. In conclusion, we described here a new experimental model of human middle ear cholesteatoma, and demonstrated that KGF and KGF receptor paracrine action play an essential role in middle ear cholesteatoma formation in an in vivo model.
Assuntos
Colesteatoma da Orelha Média/genética , DNA/genética , Fator 7 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Animais , Colesteatoma da Orelha Média/metabolismo , Modelos Animais de Doenças , Fator 7 de Crescimento de Fibroblastos/biossíntese , Humanos , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The surface of the middle ear is composed of the tympanic membrane (TM) and the middle ear mucosa (MEM). A number of diseases and conditions such as otitis media, middle ear cholesteatoma, and perforation of the TM have been reported to cause dysfunction of the middle ear, ultimately leading to high-frequency hearing loss. Despite its importance in repairing the damaged tissues, the stem/progenitor cells of the TM and the MEM epithelia remains largely uncharacterized due, in part, to the lack of an optimal methodology to expand and maintain stem/progenitor cells long-term. Here, we show that suppression of TGF-ß signaling in a low Ca2+ condition enables long-term proliferation of p63-positive epithelial stem/progenitor cells of the TM and the MEM while avoiding their malignant transformation. Indeed, our data show that the expanded TM and MEM stem/progenitor cells respond to Ca2+ stimulation and differentiate into the mature epithelial cell lineages marked by cytokeratin (CK) 1/8/18 or Bpifa1, respectively. These results will allow us to expand epithelial stem/progenitor cells of the TM and MEM in quantity for large-scale analyses and will enhance the use of mouse models in developing stem cell-mediated therapeutic strategies for the treatment of middle ear diseases and conditions.
Assuntos
Perfuração da Membrana Timpânica , Membrana Timpânica , Camundongos , Animais , Orelha Média , Células-Tronco , Mucosa , Fator de Crescimento Transformador beta , Proliferação de Células , Perfuração da Membrana Timpânica/etiologia , Glicoproteínas , FosfoproteínasRESUMO
HYPOTHESIS: To evaluate the effectiveness of the menin-MLL inhibitor, MI503, as a conservative treatment of middle ear cholesteatoma (cholesteatoma) in a mouse model and to confirm its safety profile regarding auditory function in vivo. BACKGROUND: Cholesteatoma is a mass formed by the keratinizing squamous epithelium in the tympanic cavity and/or mastoid and subepithelial connective tissue and by the progressive accumulation of keratin debris with/without a surrounding inflammatory reaction. Although the main treatment is surgical therapy, the techniques to prevent recurrence remain a critical area of research. Recently, the use of MI503 in experiments resulted in the inhibition of the growth of cholesteatoma in vivo under histone modification. METHODS: After cholesteatoma was induced in ICR mice (n = 7) by keratinocyte growth factor expression vector transfection, MI503 (50 µM) or phosphate-buffered saline was topically injected for 14 days. The effects of MI503 against cholesteatoma were analyzed by micro-computed tomography images. For the in vivo ototoxicity study, a single intratympanic injection of MI503 (50 or 500 µM) or phosphate-buffered saline (n = 4 each) was done in the ICR mice. An auditory brainstem response was performed at days 0, 1, and 14. For morphological analysis, immunostaining for Phalloidin/F-actin and Myo7a was performed. RESULTS: MI503 reduced keratinocyte growth factor-induced cholesteatoma in vivo (4 of 4 [100%]). No difference was found in the mean variation of the average of the auditory brainstem response thresholds between the three groups in the in vivo ototoxicity study, thus confirming its safety profile regarding auditory function. MI503 does not demonstrate any deleterious effects on murine hair cells when assessed by immunostaining. CONCLUSION: These findings demonstrate an encouraging safety profile for the use of menin-MLL inhibitor for the conservative treatment of cholesteatoma.
Assuntos
Colesteatoma da Orelha Média , Colesteatoma , Ototoxicidade , Animais , Camundongos , Colesteatoma da Orelha Média/tratamento farmacológico , Fator 7 de Crescimento de Fibroblastos , Epigênese Genética , Camundongos Endogâmicos ICR , Microtomografia por Raio-X , Colesteatoma/cirurgia , Orelha Média , FosfatosRESUMO
BACKGROUND: External auditory canal (EAC) squamous cell carcinoma (SCC) is a rare disease, and the survival rate is low in the advanced stages. It has been reported that miR-34a expression is low in many cancers and acts as a tumor suppressor, but its function in EACSCC has not yet been reported. AIMS: To analyze the miR-34a expression levels in EACSCC specimens using in situ hybridization (ISH). MATERIAL AND METHODS: We performed microRNA ISH for miR-34a detection and immunohistochemical analysis of p53 and Ki67 in the EACSCC and otitis externa (OE) specimens. RESULTS: miR-34a was expressed in the basal and suprabasal layers in the OE epidermis. The pronounced expression of miR-34a was observed in the two cases of T2 (Stage II). In the one case of T3 (Stage III), it was almost the same as that of the OE. On the other hand, the expression levels of miR-34a in the one case of T3 (Stage IV) and two cases of T4 (Stage IV) were apparently reduced. CONCLUSION: We demonstrated that the expression level of miR-34a was higher in early-stage EACSCC and lower in advanced-stage EACSCC. SIGNIFICANCE: The expression level of miR-34a may predict a prognosis in patients with advanced-stage EACSCC.
Assuntos
Carcinoma de Células Escamosas , MicroRNAs , Carcinoma de Células Escamosas/patologia , Meato Acústico Externo/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , PrognósticoRESUMO
Objectives: The epidermal growth factor receptor (EGFR) is related to the invasion and metastasis of external auditory canal (EAC) squamous cell carcinoma (SCC). The phosphoinositide-dependent protein kinase-1 (PDPK1) accelerates tumor cell growth through anti-apoptotic signaling under the influence of downstream EGFR-mediated signaling pathways. In this study, we investigated the EGFR/PDPK1 axis in the EAC under EGF stimulation. Methods: We confirmed EGFR and PDPK1 expression in human EACSCC specimens immunohistochemically. We next transfected the EGF expression vector in the mouse EAC and then conducted a PDPK1 inhibitory experiment. Immunohistochemical analysis was performed in the mouse EAC, using anti-EGF, anti-EGFR, anti-PDPK1, and anti-Ki67 antibodies. Immunohistochemical analysis of cleaved caspase-3 and terminal deoxy(d)-UTP nick end labeling (TUNEL) detection assays were also performed for the assessment of apoptosis in the inhibitory experiment. Results: Immunohistochemical analysis revealed overexpression and colocalization of EGFR and PDPK1 in human EACSCC specimens. The growth of a protuberant tumor was observed in the mouse EAC in which EGF expression vector was transfected, and EGF, EGFR, PDPK1, and Ki67 labeling indexes (LIs) were significantly increased. PDPK1 inhibition then induced normal epithelial appearance in the EAC. Moreover, EGF, EGFR, PDPK1, and Ki67 LIs were decreased, and cleaved caspase-3 and TUNEL LIs were increased in the EAC. Conclusion: We demonstrated the possibility that PDPK1 plays an important role in EACSCC.Level of Evidence: NA.
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Middle ear cholesteatoma is characterized by enhanced proliferation of epithelial cells with aberrant morphological characteristics. To investigate the origin of the cholesteatoma cells, we analyzed spontaneously occurring cholesteatomas associated with a new transplantation model in Mongolian gerbils (gerbils). Cholesteatomas were induced in gerbils with a transplanted tympanic membrane by using the external auditory canal (EAC) ligation method. After the pars flaccida of the tympanic membranes were completely removed from male gerbils, corresponding portions of tympanic membranes of female gerbils were transplanted to the area of defect, and then we ligated the EAC (hybrid-model group). As a control group, the EAC of normal male and female gerbils was ligated without myringoplasty. In all ears of each group, the induced cholesteatomas were seen. In situ PCR was then performed to detect the mouse X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene on the paraffin sections. One pgk-1 spot in the epithelial nuclei was detected in male cholesteatoma, and two pgk-1 spots were detected in female cholesteatoma, respectively. On the other hand, in the hybrid-model group, we detected not only one but also two pgk-1 spots in the epithelial nuclei of cholesteatoma. These results strengthened the evidence that the origin of epithelial cells in cholesteatoma is the tympanic membrane in this model, but not the residential middle ear epithelial cells or the skin of the EAC.
Assuntos
Colesteatoma da Orelha Média , Modelos Animais de Doenças , Gerbillinae , Animais , Colesteatoma da Orelha Média/etiologia , Colesteatoma da Orelha Média/patologia , Meato Acústico Externo/cirurgia , Células Epiteliais/metabolismo , Feminino , Masculino , Camundongos , Fosfoglicerato Quinase/genética , Reação em Cadeia da Polimerase , Membrana Timpânica/patologia , Membrana Timpânica/transplanteRESUMO
Middle ear acquired cholesteatoma is a pathological condition associated with otitis media, which may be associated with temporal bone resorption, otorrhea and hearing loss, and occasionally various other complications. Cholesteatoma is characterized by the enhanced proliferation of epithelial cells with aberrant morphologic characteristics. Unfortunately, our understanding of the mechanism underlying its pathogenesis is limited. To investigate its pathogenesis, different animal models have been used. This paper provides a brief overview of the current status of research in the field of pathogenesis of middle ear acquired cholesteatoma, four types of animal models previously reported on, up-to-date cholesteatoma research using these animal models, our current studies of the local hybrid ear model, and the future prospect of new animal models of middle ear cholesteatoma.
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Colesteatoma da Orelha Média/patologia , Modelos Animais de Doenças , Animais , Derme/patologia , Orelha Média/patologia , Implantes ExperimentaisRESUMO
Distinct histone modifications regulate gene expression in certain diseases, but little is known about histone epigenetics in middle ear cholesteatoma. It is known that histone acetylation destabilizes the nucleosome and chromatin structure and induces gene activation. The association of histone acetylation with chronic inflammatory diseases has been indicated in recent studies. In this study, we examined the localization of variously modified histone H3 acetylation at lysine 9, 14, 18, 23, and 27 in paraffin-embedded sections of human middle ear cholesteatoma (cholesteatoma) tissues and the temporal bones of an animal model of cholesteatoma immunohistochemically. As a result, we found that there was a significant increase of the expression levels of H3K27ac both in human cholesteatoma tissues and the animal model. In genetics, super-enhancers are clusters of enhancers that drive the transcription of genes involved in cell identity. Super-enhancers were originally defined using the H3K27ac signal, and then we used H3K27ac chromatin immunoprecipitation followed by sequencing to map the active cis-regulatory landscape in human cholesteatoma. Based on the results, we identified increased H3K27ac signals as super-enhancers of the FOXC2 loci, as well as increased protein of FOXC2 in cholesteatoma. Recent studies have indicated that menin-MLL inhibitor could suppress tumor growth through the control of histone H3 modification. In this study, we demonstrated that the expression of FOXC2 was inhibited by menin-MLL inhibitor in vivo. These findings indicate that FOXC2 expression under histone modifications promoted the pathogenesis of cholesteatoma and suggest that it may be a therapeutic target of cholesteatoma.
Assuntos
Colesteatoma da Orelha Média/metabolismo , Epigênese Genética/genética , Fatores de Transcrição Forkhead , Histonas/metabolismo , Animais , Epigenômica , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Camundongos Endogâmicos BALB CRESUMO
OBJECTIVE: Cholesteatoma is an epithelial lesion that expands into the middle ear, resulting in bone destruction. The acceleration of the proliferative activity of epithelial stem/progenitor cells is involved in the pathogenesis of cholesteatoma. Recently, the use of a menin-mixed lineage leukemia 1 (MLL1) inhibitor, MI503, in experiments has resulted in inhibition of the growth of tumors under histone modification. In this study, we investigated the effects of the menin-MLL inhibitor against cholesteatoma growth in an in vivo model. METHODS: We first correlated the expression level of histone H3 trimethylation at lysine 4 (H3K4me3) among cholesteatoma cases, chronic otitis media cases and normal skin tissues. Based on the role of keratinocyte growth factor (KGF) in the development of cholesteatoma, KGF-expression vector was transfected into the ear and we analyzed the expression level of H3K4me3. After cholesteatoma was induced, MI503 was administered daily into the ear for 14 days. RESULTS: We detected the highest labeling index of H3K4me3 in the cholesteatoma specimens. After KGF-expression vector transfection in the mouse ear, a high expression level of H3K4me3 was observed in the epithelial layers. The use of MI503 reduced cholesteatoma in the in vivo model and decreased the proliferation of epithelial stem/progenitor cells in a dose-dependent manner. CONCLUSION: We demonstrated that inhibition of the menin-MLL interaction may be a potentially useful strategy in the conservative treatment of cholesteatoma.
Assuntos
Colesteatoma da Orelha Média , Animais , Orelha Média , Camundongos , Otite Média , Células-Tronco , TransfecçãoRESUMO
PURPOSE OF REVIEW: Middle ear cholesteatoma is an epithelial lesion that expands into the middle ear, resulting in bone destruction. However, the pathogenesis of this has been unknown. The purpose of this review is to understand the role of keratinocyte growth factor (KGF) during epithelial stem and/or progenitor cell proliferation in middle ear cholesteatoma. RECENT FINDINGS: Many researchers have investigated the molecular mechanism of middle ear cholesteatoma to establish a conservative treatment. Recently, some studies have focused on the stem cells of middle ear cholesteatoma and their detection, but the key molecules for stem cell formation were not shown. SUMMARY: We established an animal model for middle ear cholesteatoma and are showing the results of our studies. KGF expression accelerates the proliferation of stem/progenitor cells through the induction of transcription factor p63 expression in the epithelium of the tympanic membrane and mucosal epithelium overlying the promontory of the cochlea and within the attic. This is typical in middle ear cholesteatoma. Moreover, the partial epithelial-mesenchymal transition under the p63 signaling pathway plays an essential role in epithelial cell growth in middle ear cholesteatoma formation. Understanding p63 expression following KGF expression and associated signaling events can improve therapeutic outcomes in patients with middle ear cholesteatoma.
Assuntos
Colesteatoma da Orelha Média/metabolismo , Colesteatoma da Orelha Média/patologia , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proliferação de Células , Colesteatoma da Orelha Média/etiologia , Transição Epitelial-Mesenquimal , Humanos , Transdução de SinaisRESUMO
OBJECTIVE: The middle ear epithelium is derived from the neural crest and endoderm, which line distinct regions of the middle ear cavity. In this study, we investigated the localization of stem/progenitor cells in the middle ear mucosa of adult mice and the effects of keratinocyte growth factor (KGF) on the cell kinetics of stem/progenitor cells in vivo. METHODS: In this study, after KGF-expression vector was transfected in the ear, two kinds of thymidine analogues, BrdU and EdU, were transferred at different time points. BrdU was detected by immunohistochemistry and EdU was detected by click chemistry. We also performed immunohistochemistry using anti-Keratin14 (K14) antibody (an undifferentiated epithelial cell marker), anti-p63 antibody (a stem/progenitor cell marker) and anti-acetylated α-tubulin antibody (a ciliated epithelial cell marker). RESULTS: A large number of EdU-positive cells were detected in the thickened mucosal epithelium of the pars flaccida and attic region at Day 1 after KGF transfection. Interestingly, in the mucosal epithelium overlying the promontory of the cochlea, many EdU-positive cells were detected. These cells were also positive for K14 and p63. The acetylated α-tubulin positive cells were reduced in the attic region at Day 1 after KGF transfection. CONCLUSION: These findings indicate that KGF over-expression may increase stem/progenitor cell proliferation in the mucosal epithelium not only within the attic which is typical in middle ear cholesteatoma, but also overlying the promontory of the cochlea.
Assuntos
Orelha Média/citologia , Células Epiteliais/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Mucosa/citologia , Células-Tronco/fisiologia , Animais , Proliferação de Células , Orelha Média/metabolismo , Fator 7 de Crescimento de Fibroblastos/genética , Queratina-14/metabolismo , Masculino , Camundongos , Mucosa/metabolismo , Transativadores/metabolismo , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
DNA methylation is an important epigenetic mechanism for cellular maintenance. However, the methylation pattern and the key molecule regulated epigenetically in oral mucosal regeneration is unclear. In this study, we generated a rat oral ulcer model and investigated the cell proliferative activities and DNA methylation patterns immunohistochemically. We also performed immunohistochemical analysis of a regulator of epithelial stem/progenitor cell differentiation in the rat model. We demonstrated immunohistochemistry using antibodies for the molecules as follows: Ki-67, a marker of cellular proliferation; 5-methylcytosine (5-mC), a marker of DNA methylation; 5-hydroxymethylcytosine (5-hmC), a marker of DNA demethylation; Dnmt1, a maintenance DNA methyltransferase; Dnmt3a and Dnmt3b, de novo DNA methyltransferases; and Wnt5a, a regulator of stem/progenitor cell differentiation. In this model, re-epithelialization was completed at Day 4 after ulceration. Regenerating mucosal hypertrophy reached a peak at Day 5 and appeared normal at Day 14. Ki-67-positive cells increased at Day 2 and returned to normal at Day 6 after ulceration. The ratio of the expression level of 5-mC to 5-hmC declined at Day 5 and returned to normal at Day 6. The expression level of Dnmt1 had not changed compared to the normal control at every time point. On the other hand, the expression levels of Dnmt3a and Dnmt3b had decreased significantly at Day 5 and returned to normal at Day 6. Moreover, Wnt5a-positive cells increased at Day 5. In conclusion, oral mucosal regeneration was strictly regulated by DNA methylation. Moreover, Wnt5a might play a critical role in oral mucosal regeneration.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Mucosa Bucal/crescimento & desenvolvimento , Úlceras Orais/patologia , Regeneração , 5-Metilcitosina/análogos & derivados , Animais , Proliferação de Células , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Imuno-Histoquímica , Ligantes , Masculino , Ratos , Ratos Wistar , Proteína Wnt-5a/metabolismoRESUMO
INTRODUCTION: Partial epithelial-mesenchymal transition (p-EMT) is a process by which epithelial cells partially lose their intercellular adhesion and change to obtain migration ability. The transcription factor p63 regulates the expression of cadherin family and induces epithelial cell proliferation. In this study, we hypothesized that p-EMT under p63 expression may be a key factor in epithelial cell growth in middle ear cholesteatoma. METHODS: Specimens were surgically excised from patients with congenital cholesteatoma (CC) (nâ=â48), acquired middle ear cholesteatoma (AC) (nâ=â120), and normal skin tissue (nâ=â34). We analyzed immunohistochemically for the EMT marker (N-cadherin), adherence junction marker (E-cadherin), and tight junction marker (claudin-1, claudin-4, occludin). We also examined the labeling index (LI) of p63 and Proliferating cell nuclear antigen (PCNA) (late S phase marker), and Snail expression as a mobility marker. RESULTS: The expression of p63 (CC 51.0â±â7.4%, AC 50.0â±â5.9%) was significantly higher in the thickened epithelium of CC and AC compared with normal skin tissue (pâ<â0.0001). The loss of E-cadherin was observed (CC 50.0%, AC 55.8%) but the expression patterns in the tight junction were almost normal. N-cadherin was partially detected in the basal and upper layer of epithelium in CC and AC. In contrast to that of normal skin tissue, the LI of PCNA was significantly higher in AC (pâ<â0.0001). The positive rate of Snail was significantly higher in CC (pâ<â0.0001). CONCLUSION: This study indicates that p-EMT via the p63 signaling pathway might plays an essential role in epithelial growth in AC and CC formation, although tight junction formation and terminal differentiation were not affected in those processes.
Assuntos
Colesteatoma da Orelha Média/patologia , Colesteatoma/congênito , Transição Epitelial-Mesenquimal/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adulto , Colesteatoma/metabolismo , Colesteatoma/patologia , Colesteatoma da Orelha Média/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To evaluate the effect of our large-sized silicone sheet upon postoperative recovery of mastoid aeration in ears after surgery including mastoidectomy and soft-wall reconstruction (SWR). STUDY DESIGN AND SETTING: Retrospective chart review in a tertiary care center. SUBJECTS AND METHODS: Recovery of mastoid aeration was assessed by CT 4 to 12 months after surgery on 72 ears (69 patients), in which the silicone sheet covering from the eustachian tube (ET) to the mastoid was placed after SWR procedure with mastoidectomy were done for their chronic otitis media. Results were compared with those with a small silicone sheet. RESULTS: Recovery of mastoid aeration was significantly better in the large-silicone-sheet group than in the small-silicone-sheet group (chi(2) value = 11.7146, P = 0.0006). CONCLUSION: This preliminary study suggested that our large-sized silicone sheet may be effective for postoperative recovery of mastoid aeration even in ears operated with SWR procedure.
Assuntos
Colesteatoma da Orelha Média/cirurgia , Processo Mastoide/cirurgia , Prótese Ossicular , Silicones , Timpanoplastia/instrumentação , Adolescente , Adulto , Idoso , Criança , Colesteatoma da Orelha Média/patologia , Estudos de Coortes , Meato Acústico Externo/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Desenho de Prótese , Estudos Retrospectivos , Resultado do TratamentoRESUMO
OBJECTIVE: To evaluate the validity of cochlear implantation (CI) on prelingually deafened adults who have been trained by auditory-verbal/oral communication since childhood. METHODS: Preoperative and postoperative data was investigated regarding the rehabilitation, hearing level, and educational experience of eight prelingually deafened adults. All eight patients were diagnosed with severe to profound sensorineural hearing loss (preoperative hearing levels were over 100 dB). All used hearing aids (HA) before the age of two and were trained by auditory-verbal/oral communication since childhood. The average age of the patients at the time of their CI operations was 23.3 ranging from 18 to 29 years of age. The average postoperative observation time was 55.4 months ranging from 11 to 90 months. RESULTS: Improvement was achieved not only on the pure-tone hearing threshold, but also in speech perception on tests using the Japanese video speech discrimination score (SDS) system. All of them now use CI very well in their daily lives and play important roles in society. CONCLUSION: It was demonstrated that even prelingually deafened adult patients could achieve considerable improvement through CI when they were trained well by auditory-verbal/oral communications since childhood. The indications of CI for prelingually deafened adults must be determined carefully, but all of them do not have to be rejected only because they are prelingually deafened. In other words, CI could be recommended for prelingually deafened adult patients if they received habilitation well with consistent auditory-verbal/oral training using well-fitted HAs.