| Objective:Cholesteatoma of the middle ear is a chronic inflammation characterized by the hyperproliferation and apoptosis of keratinizing squamous epithelium in the middle ear cavity and destruction of adjacent bone. Although its pathogenesis is not clear yet, recent researches show that cytokines plays an important role in the cause and development of cholesteatoma. The proliferative activity of experimental middle ear cholesteatoma, aggravated bone destruction and an excessive accumulation of keratin debris as well as chromosomal abnormalities, changes in signal transduction have direct or indirect links with cytokines. Therefore, the depth researches of cytokines in the specific role of acquired cholesteatoma are becoming important, which is a key ring in research clearly acquired cholesteatoma pathogenesis. This study in conducted to explore tumour necrosis factor receptor 1(TNFR1), caspase-8 in acquired human middle ear cholesteatoma, to discuss the expression and activation of nuclear factor-κB (NF-κB) in acquired human middle ear cholesteatoma and their roles in the bone destruction of acquired human middle ear cholesteatoma.Methods:1. Immunohistochemical two-step method is utilized to check out the expression of TNFR1, caspase-8, NF-κB, proliferation cell nuclear antigen (PCNA) in 36 cases of the acquired human middle ear cholesteatoma epithelium specimens, 20 cases of auditory meatal skin samples of cholesteatoma and 15 cases of normal auditory meatal skin.2. Through Western Blot, the protein expression of TNFR1, caspase-8 are determined in the 25 cases of acquired human middle ear cholesteatoma epithelium specimens, 25 cases of auditory meatal skin samples of cholesteatoma and 15 cases of normal auditory meatal skin. 3. The expression of Apoptotic cells is determined by TUNEL technique in the 36 cases of acquired human middle ear cholesteatoma epithelium specimens, 20 cases of auditory meatal skin samples of cholesteatoma and 15 cases of normal auditory meatal skin.Results:1. The positive expression of TNFR1, caspase-8, NF-κB, PCNA were found in acquired human middle ear cholesteatoma, with positive rates of ( 68.61±5.74)%,(8 0.14±4.79)%,(5 0.67±4.44)% and ( 77.03±4.33)% respectively. TNFR1, caspase-8 were mainly expressed in membrane and cytoplasm of cells, NF-κB and PCNA were mainly expressed in cell nucleus. In cholesteatoma tissue, TNFR1, caspase-8, NF-κB, PCNA were mainly found in the spinous layer, granular layer and basal layer in cholesteatoma with weak expression in the stratum corneum. While in the auditory meatal skin samples of cholesteatoma and the normal auditory meatal skin, TNFR1, caspase-8, NF-κB and PCNA positive cells were found decreasing drastically and distributed mainly in the spinous layer and granular layer with weak expression in the basal layer and the stratum corneum. The expression of TNFR1, caspase-8, NF-κB, PCNA in cholesteatoma tissues was higher than that in auditory meatal skin samples of cholesteatoma and in normal auditory meatal skin ( p <0.05). While the expression of TNFR1, caspase-8, NF-κB, PCNA did not show a significant difference between the auditory meatal skin samples of cholesteatoma and the normal auditory meatal skin ( p >0.05).2. The positive expression of apoptosis cells was found in cholesteatoma, auditory metal skin of cholesteatoma and normal human auditory metal skin with respective positive rates of ( 75.67±4.58)%,(3 0.52±6.93)% and (3 1.47±6.36)%. Apoptotic cells were mainly found in the spinous layer, granular layer and basal layer. The cell apoptotic rates in acquired human middle ear cholesteatoma were significantly higher than in auditory meatal skin samples of cholesteatoma and normal auditory meatal skin ( p <0.05). Expression of apoptotic cells did not show a significant difference between the auditory meatal skin samples of cholesteatoma and the normal auditory meatal skin ( p >0.05).3. The expression of TNFR1, caspase-8 protein in acquired human middle ear cholesteatoma was significantly higher than that in auditory meatal skin samples of cholesteatoma. There was no expression of TNFR1, caspase-8 protein in the normal auditory meatal skin. TNFR1, caspase-8 protein was located in 55KD, 55.3KD respectively. 4. There was notable relationship between the expression of PCNA and the activity of NF-κB and caspase-8 in acquired human middle ear cholesteatoma ( p <0.05).Conclusions:1. The expression and protein level of TNFR1 in acquired human middle ear cholesteatoma is greatly higher than that in auditory meatal skin samples of cholesteatoma and in normal auditory meatal skin, which indicates TNFR1 plays an important role in the pathogenetic mechanism of acquired human middle ear cholesteatoma.2. The expression of caspase-8 and apoptotic cells in acquired human middle ear cholesteatoma is noticeably higher than that in auditory meatal skin samples of cholesteatoma and in normal auditory meatal skin, which indicates caspase-8 plays an important role in the deregulation of apoptosis in keratinocyte of cholesteatoma.3. The expression of PCNA and the activity of NF-κB in acquired human middle ear cholesteatoma are significantly higher than that in auditory meatal skin samples of cholesteatoma and in normal auditory meatal skin, which indicats NF-κB plays a key role in the hyperproliferation of cholesteatoma.4. TNFR1 may activate these two signal transmission paths of NF-κB and caspase-8, leading to the hyperproliferation and hyperapoptosis of keratinizing epithelium. Increased cell proliferation and apoptosis contribute to the accumulation of keratinous debris, which causes bone destruction. |
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