OBJECTIVETo investigate whether the cholesteatoma from different positions have different biological characteristics.

METHODSThe expression of the Ki-67 and collagen IV in 19 specimens of cholesteatoma were stained immunohistochemically using the SP method, according to the origin of the specimens, include 7 cases from the epitympanum, 8 cases from tympanic sinus and 4 cases from the out acoustic canal. According to the severity of the inflammation in the perimatrix, the inflammation group included 7 cases, the non-inflammation group included 8 cases.

RESULTSThe average count was the same between the cholesteatoma from epitympanum and tympanic sinus. And the count of the cholesteatoma in the middle ear was also the same to the cholesteatoma from the out acoustic canal. But even from the same sample, the cholesteatoma from the positions with severe inflammation in the perimatrix count much higher, and the difference was statistically significant. Collagen IV had been found to localized in the basic membrane. In some specimens the staining of the collagen IV was not continuous.

CONCLUSIONSThe results suggested that it was the severity of inflammation in the perimatrix influenced the differences, but not the origins of the cholesteatoma.

Adolescent ; Adult ; Aged ; Cholesteatoma ; classification ; metabolism ; pathology ; Cholesteatoma, Middle Ear ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; metabolism ; Male ; Middle Aged ; Young Adult

OBJECTIVETo study various expressions and roles of MMP-2,9 in cholesteatoma from different positions.

METHODSMMP-2 and MMP-9 were detected with immunohistochemical methods using antibody in 18 attic cholesteatoma, 16 tympanic sinus cholesteatoma, 10 auditory meatal skin in cholesteatoma (CAMS) and 10 normal auditory meatal skin (NAMS).

RESULTSThe stain in cholesteatoma was stronger than that in CAMS and NAMS (P < 0.05). Expression of MMP-2,9 was seen in all layers of the epithelium in cholesteatoma and the stain was strongest in basement membrane. Expression of MMP-2,9 in tympanic sinus cholesteatoma was stronger than that in attic cholesteatoma. The expression of MMP-2 and MMP-9 were closely related in cholesteatoma (r = 0.974, P < 0.01). The expression of MMP-2,9 in cholesteatoma was related with destruction of ossicular chain (r = 0.789, P < 0.01; r = 0.803, P < 0.01).

CONCLUSIONSMMPs play a very important role in bone destruction in cholesteatoma. The destruction of bone in tympanic sinus cholesteatoma is more severe than that in attic cholesteatoma. Tympanic sinus cholesteatomas are more invasive in destruction surround bone matrix.

Adolescent ; Adult ; Aged ; Cholesteatoma, Middle Ear ; metabolism ; pathology ; Ear, Middle ; pathology ; Female ; Humans ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; Skin ; pathology ; Young Adult

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

Aural cholesteatoma is characterized by invading squamous epithelia with altered growth properties. Cytokeratin (CK) expression is affected in epidermal proliferative diseases and represents the alterations of keratinocyte proliferation, differentiation, and migration. In the present study, the intensity of CK immuno-expression was determined, using densitometry at various sites in experimental cholesteatoma in order to characterize changes of keratinocytes. With cholesteatoma formation, CK4, a marker for non-keratinizing epithelia, increased in the suprabasal layers of the annular external auditory canal (EAC) and at the pars tensa indicating an altered differentiation and migration of keratinocytes. CK5/6, a marker of keratinizing squamous epithelium, increased only at the pars tensa of the tympanic membrane, indicating basal keratinocyte hyperplasia. CK1/10 increased in the suprabasal layer at the annular EAC, and at the peripheral pars tensa, indicating increased terminal differentiation of keratinocytes. CK13/16, markers of differentiation and hyperproliferation, increased in suprabasal layer of the EAC, and at the peripheral pars tensa. However, it decreased in the basal layer of the EAC, indicating hyperproliferation and migration of keratinocytes. The findings of this study support the basal cell hyperplasia hypotheses for the pathogenesis of aural cholesteatoma, with regard to hyperproliferation, migration, and an altered differentiation of keratinocytes.
Animals ; Biological Markers ; Cell Division ; Cell Movement ; Cholesteatoma, Middle Ear/*metabolism/*pathology ; Densitometry ; Gerbillinae ; Keratinocytes/metabolism/pathology ; Keratins/*biosynthesis

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 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

OBJECTIVETo explore the expression and activation of NF-kappaB in middle ear cholesteatoma.

METHODSThe protein expression of NF-kappaB p65 in 21 middle ear cholesteatoma tissues and 8 normal external ear canal skin obtained in middle ear surgery were examined by immunohistochemistry; NF-kappaB DNA binding activity in these two kinds of tissues were also detected by electrophoretic motility shift assay (EMSA). The influence of cholesteatoma debris on the NF-kappaB DNA binding activity of HaCat cell were further analyzed.

RESULTSAll epithelial cell of cholesteatoma revealed a relatively abundant plasma expression of NF-kappaB p65 protein, among which 12 cases showed nuclear positive expression. In contrast,the normal skin epithelium only revealed a sparse plasma distribution of NF-kappaB protein. The levels of NF-kappaB p65 protein expression in the epithelium of middle ear cholesteatoma tissue and normal skin were 0.168 +/- 0.051, 0.088 +/- 0.019 (t = 4.211, P < 0.01), respectively. The NF-kappaB DNA binding activities of cholesteatoma [(16.5 +/- 10.1)%] were also higher than those in normal skin [(1.38 +/- 1.24)%, t = 3. 600, P = 0.014]. The NF-kappaB DNA binding activity of HaCat cell increased when exposed to cholesteatoma debris in a dose dependent manner.

CONCLUSIONNF-kappaB might be an important factor which was involved in the occurrence and development of cholesteatoma.

Adolescent ; Adult ; Biopsy ; Case-Control Studies ; Child ; Cholesteatoma, Middle Ear ; metabolism ; pathology ; Electrophoretic Mobility Shift Assay ; Female ; Humans ; Male ; Middle Aged ; NF-kappa B ; metabolism ; Young Adult

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

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