OBJECTIVE: In order to investigate the interaction between the cytokines and keratinocyte and determine the role of cytokines in hyperproliferative of chronic otitis media with cholesteatoma, we observe the expression of matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and its receptor (KGFR) in middle ear cholesteatoma. METHOD: We examined the expression of MMP9, VEGF, KGF, KGFR and Ki-67 by immunohistochemistry in 50 specimens from chronic otitis media with cholesteatoma and 15 specimens from the normal skin of external auditory meatus. Ki-67 as an evaluation of cholesteatoma proliferation markers were used to detect the keratinocyte proliferative activity. RESULT: (1) The expression of VEGF and MMP9 in cholesteatoma specimens was higher than normal skin, and the difference was statistically significant (t = 4.914, P < 0.01; t = 3.284, P < 0.01). (2) The expression of KGF and KGFR in middle ear tissues was higher than normal skin, and the difference was statistically significant (t = 4.814, P < 0.01; t = 3.104, P < 0.01); The expression of KGF and KGFR increased, and the expression of Ki-67 also correspondly increased in the cholesteatoma. (3) In the tissue MMP9 and VEGF were positive. Mean optical density increased as well. KGF expression also increased accordingly. CONCLUSION MMP9, VEGF, KGF and KGFR proteins played an important role in hyperproliferation of cholesteatoma tissues. VEGF, MMP9 and KGF had a synergistic effect in hyperproliferation of cholesteatoma tissues.
Cholesteatoma, Middle Ear ; pathology ; Cytokines ; metabolism ; Ear Canal ; metabolism ; Ear, Middle ; metabolism ; Fibroblast Growth Factor 7 ; metabolism ; Humans ; Immunohistochemistry ; Keratinocytes ; cytology ; Ki-67 Antigen ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Otitis Media ; pathology ; Receptor, Fibroblast Growth Factor, Type 2 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.
Animals ; Ear, Middle/*metabolism/pathology ; Mice ; Microscopy, Electron, Scanning ; Mucin 5AC/genetics/*metabolism ; RNA, Messenger/*genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Spectrophotometry

PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.
Animals ; Ear, Middle/*metabolism/pathology ; Mice ; Microscopy, Electron, Scanning ; Mucin 5AC/genetics/*metabolism ; RNA, Messenger/*genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Spectrophotometry

OBJECTIVE: Detect the expressions of the protein tyrosine phosphatase gene (PTEN), phosphorylated protein kinase B (P-AKT) and phosphorylated extracellular signal-regulated kinase (P-ERK) in human middle ear cholesteatoma tissue and its correlation to explore their important role in the mechanism of the formation of cholesteatoma. METHOD: Use immunohistochemical SABC method (SABC immunohistochemical method) to detect the expressions and location of PTEN, P-AKT and P-ERK proteins in 40 cases of middle ear cholesteatoma tissue samples and 15 cases of normal ear skin specimens. Use Western blot to detect the expression levels of PTEN, P-AKT, P-ERK proteins and the internal reference GAPDH in 20 cases of cholesteatoma tissue and 10 cases in nor mal ear skin specimens. RESULT: (1) Immunohistochemistry showed coloring of PTEN both in the nucleus and cytoplasm of cholesteatoma and normal skin . Nuclear PTEN positive expression rates in the cholesteatoma was significantly lower than normal skin, and the difference was significant (P < 0.01); cytoplasm PTEN positive expression rates in the cholesteatoma was significantly lower than normal skin, and the difference was significant (P < 0.01); P-AKT mainly expresses in the cytoplasm of cholesteatoma and normal skin. The positive expression rates in the cholesteatoma was significantly higher than normal skin,and the difference was significant (P < 0.01); the P-ERK mainly colors in cholesteatoma and normal skin cell nucleus. the positive expression rates in the cholesteatoma was significantly higher than normal skin, and the difference was significant (P < 0.01). In cholesteatoma specimens, there was a significantly negative relationship (P < 0.01) between PTEN, respectively, and P-AKT, P-ERK protein. (2) Western blot discovered: the expression of PTEN in cholesteatoma was significantly less than the amount of expression in normal skin; P-AKT and P-ERK expression in cholesteatoma was significantly more than the level in normal skin. CONCLUSION Abnormal expression of PTEN, P-AKT and P-ERK protein in cholesteatoma may be closely related to antiapoptosis and high degree of proliferation in cholesteatoma. Expression of PTEN deletion leads to the weakening of the inhibition. Excessive expression of P-AKT gives rise to cholesteatoma epithelial cell apoptosis inhibited; excessive PERK expression result to cholesteatoma epithelial cell proliferation strengthened.
Apoptosis ; Cell Proliferation ; Cholesteatoma, Middle Ear ; metabolism ; pathology ; Epithelium ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; PTEN Phosphohydrolase ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism

OBJECTIVE: To investigate the expression and concentration of lipopolysaccharide (LPS) and matrix metalloproteinase-9 (MMP-9) in middle ear cholesteatoma and discuss their relations. METHOD: Twenty-nine cases of middle ear cholesteatoma tissue, 18 cases of external auditory canal tissue were detected by limulus amebocyte lysate assay (LAL-assay), and expression of MMP-9 protein in formalin-fixed, paraffin-embedded tissues was detected by immunohistochemical method. RESULT: The concentrations of LPS in cholesteatoma were higher than that in external auditory canal tissues. In group of cholesteatoma: M = 0.739 0, IQR = 0.6203, and in group of external auditory canal tissues: M = -0.2538, IQR = 1.1692 (P < 0.01). In cholesteatoma groups, in extensive type: M = 0.8403, IQR = 0.5254; in localized type: M = 0.4048, IQR = 0.6139, the concentrations of LPS were higher in extensive cholesteatoma in comparison with localized cholesteatoma (P < 00.05). In cholesteatoma epithelium samples, MMP-9 were 79.3%. Compared with external auditory canal epithelium, the expression of MMP-9 was higher in middle ear cholesteatoma epithelium (P < 0.05). There was no significant difference in the expression of MMP-9 between two types of cholesteatoma epithelium (P > 0.05). LPS, MMP-9 weren't significantly correlated by Spearman test. CONCLUSION LPS was responsible for middle ear cholesteatoma and its related bone erosion. MMP-9 was related to the development of middle ear cholesteatoma. There's no correlation between LPS and MMP-9.
Adult ; Cholesteatoma, Middle Ear ; metabolism ; pathology ; Female ; Humans ; Lipopolysaccharides ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Receptors, Calcitriol ; metabolism

OBJECTIVE: To investigate the expression and clinical significance of MMP9 and MVD in the carcinogenesis of squamous cell carcinoma of external auditory canal and middle ear. METHOD: Immunohistochemical SP method was used to detect the expression of MMP9 and MVD proteins in 26 squamous cell carcinoma tissues of external auditory canal and middle ear and 20 normal external ear canal skin tissues. RESULT: The positive rate of MMP9 in squamous cell carcinoma tissues of external auditory canal and middle ear was 73.1% (19/26) lower than that in the normal external ear canal skin tissues 25.0% (5/20). The positive rates of CD34 were 33.58 +/- 3.04 and 22.50 +/- 5.22, respectively. The positive rates of MMP9 and CD34 were correlated with the histological grade and tumor grade, but had no relationship with age and sex. The positive rates between MMP9 and CD34 were related (r=0.42, P<0.05). CONCLUSION MMP9 may be involved in the carcinogenesis of squamous cell carcinoma of external auditory canal and middle ear, and may play an important role in the invasion and metastasis of squamous cell carcinoma of external auditory canal and middle ear. MMP9 and CD34 play a cooperative role in the process of squamous cell carcinoma of external auditory canal and middle ear.
Adult ; Aged ; Antigens, CD34 ; metabolism ; Carcinoma, Squamous Cell ; blood supply ; metabolism ; Ear Canal ; metabolism ; Ear Neoplasms ; blood supply ; metabolism ; Ear, Middle ; metabolism ; Female ; Humans ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Microvessels ; pathology ; Middle Aged ; Neovascularization, Pathologic

Adenoma ; metabolism ; pathology ; Adult ; Carcinoma, Renal Cell ; metabolism ; pathology ; Diagnosis, Differential ; Ear Neoplasms ; complications ; genetics ; metabolism ; pathology ; surgery ; Endolymphatic Sac ; Female ; Follow-Up Studies ; Humans ; Kidney Neoplasms ; metabolism ; pathology ; Middle Aged ; Paraganglioma ; metabolism ; pathology ; Point Mutation ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; von Hippel-Lindau Disease ; complications ; genetics ; metabolism

OBJECTIVE: To detect the expression and localization of macrophages and BMP2 (bone morphogenetic protein 2) in the middle ear mucosa of TS, to explore its role in the pathogenesis of TS. METHOD: Seventeen patients with TS were studied and 17 matching cases with simple chronic suppurative otitis media (COM) were enrolled as control. Immunohistochemistry was performed to detect the expression and localization of CD68 (the symbol of macrophage) and BMP2 in the middle ear mucosa. And the expression difference in the TS and COM were analyzed. RESULT: Positive cells of CD68 was mainly localized in the submucosa layer. Each group had 12 cases with positive expression of CD68 (70.59%) and had no difference in the number of positive cells (TS: 6.94 +/- 6.08; COM: 7.59 +/- 7.84; P=0.79). All the specimens had positive expression of BMP2 and BMP2 protein was expressed in the mucosa layer and submucosa layer. There was statistical difference in the number of BMP2 expression between TS and COM group. In TS group, all the sclerotic plaques were surrounded with macrophages and BMP2 positive cells. The cells surrounding sclerotic plaques were synchronously expressing CD68 positive and BMP2 positive. CONCLUSION Macrophages should take part in the pathogenesis of TS. BMP2 secreted by macrophage was important in formation of TS.
Adolescent ; Adult ; Bone Morphogenetic Protein 2 ; metabolism ; Ear Diseases ; metabolism ; pathology ; Ear, Middle ; metabolism ; pathology ; Female ; Humans ; Macrophages ; metabolism ; Male ; Middle Aged ; Sclerosis ; metabolism ; pathology ; Young Adult

OBJECTIVE: To investigate the expression of Hypoxia inducible factor-1alpha (HIF-1alpha) and inducible Nitric Oxide Synthase (iNOS) in cholesteatoma and approach the possible role of them in the formation and development of the middle ear cholesteatoma. METHOD: Immunohistochemical SP method was used to examine the expression of HIF-1alpha and iNOS protein in 21 middle ear cholesteatoma specimens and 11 samples of normal external ear canal skin. RESULT: In the 21 middle ear cholesteatoma, in epithelium tissue samples and 11 external ear channel's normal skin specimens, the expression index of HIF-1alpha was 52.49 +/- 13.80, 0.60 +/- 0.49, and that of iNOS was 92.05 +/- 27.84, 1.15 +/- 0.84 respectively. The HIF-1alpha and iNOS expression index of cholesteatoma epithelium was significantly higher than that of external ear channel's normal skin (P < 0.01). Moreover in the cholesteatoma epithelium, linear correlation analysis showed that iNOS protein was positively correlated with HIF-1alpha protein expression (r = 0.536, P < 0.05). CONCLUSION High density of HIF-1alpha and iNOS are found in the epithelium of cholesteatoma; Hypoxia and its relative factors HIF-1alpha,iNOS may play an important role in the formation and development of cholesteatoma.
Cholesteatoma, Middle Ear ; metabolism ; pathology ; Epithelium ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Nitric Oxide Synthase Type II ; metabolism

OBJECTIVE: To investigate the expressions of nuclear factor-kappa B ligand (RANKL), receptor activator of nuclear factor-kappa B(RANK)and osteoprotegerin (OPG) and the relation of RANKL with bone- erosion in human cholesteatoma tissue. METHOD: Thirty cholesteatoma and twenty normal auditory canal skin specimens were investigated. The expressions of RANKL, RANK and OPG were examined by immunohistochemistry. RESULT: The overexpressions of the cytokine RANKL and RANK were found in infiltrated lymphocytes in the cholesteatoma tissue comparing with normal external meatal skin( t = 7. 758,6. 482, P <0. 05); While, the expression of OPG was significantly higher in cholesteatoma tissue comparing with normal external meatal skin. the OPG/ RANKL was decreased in cholesteatoma tissue comparing with normal external meatal skin( t = 8. 183, P < 0. 05). CONCLUSION This study revealed that the expressions level of RANKL and RANK were markedly increased in the perimatrix of cholesteatoma, which is closely related to the bone- erosion induced by cholesteatoma.
Adolescent ; Adult ; Case-Control Studies ; Cholesteatoma, Middle Ear ; metabolism ; pathology ; Ear Ossicles ; pathology ; Female ; Humans ; Male ; Middle Aged ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; Young Adult