| Objective:In this study,using the fact that middle ear cholesteatoma was characterized by hyperkeratinized squamous epithelium proliferation and progressive destruction of adjacent bone as a entry point.By detecting the expressions of COX-2 and PTEN in middle ear cholesteatoma to analyze their effect and correlation in the occurrence and development of middle ear cholesteatoma.Methods: 1.The western blot method: The fresh specimens of patients who had Mastoidectomy and ventriculostomy for middle ear cholesteatoma or chronic suppurative otitis media were collected from May 2018 to December 2018 in Department of Otolaryngology-Head and Neck Surgery of the Affiliated Hospital of Southwest Medical University and were saved in the refrigerator of the-80 ° C.A total of 35 cases of middle ear cholesteatoma and 20 cases of middle ear granulation were collected.The control group was 10 cases of normal external auditory canal skin which were randomly obtained from the patients during surgery.Using the Western blot method to detect the expression of COX-2 and PTEN in the three groups.The Image J Image analysis software was used to measure the relative gray value results of the three groups.SPSS22.0 statistical software was used for comparisons,and Pearson correlation analysis was used for COX-2 and PTEN.All the results were considered as statistically significant with P < 0.05.2.Immunohistochemical experiment: The fresh specimens of patients were collected during the same period mentioned above and were fixed with 10% Neutral formalin for 24 hours.All specimens were made for paraffin blocks,in which there were 20 cases of middle ear cholesteatoma paraffin block,10 cases of middle ear granulation paraffin block and 10 cases of normal external auditory canal skin paraffin blocks.The localization and quantitative analysis of COX-2 and PTEN in three groups were detected by immunohistochemistry.Image pro plus software was used to determine the average optical density of the immunohistochemical staining images.SPSS22.0 statistical software was used for comparisons.Pearson correlation analysis was used between COX-2 and PTEN.Results: 1.Westerrn blot detection results: gray values of the bands in the three groups were determined by Image J Image analysis software.COX-2 expression in the middle ear cholesteoma group,the middle ear granulation group and the normal external auditory canal skin group decreased successively,and the relative gray values in the three groups were 0.8019±0.2323、0.6432±0.1965 and 0.4518±0.1342.Respectively comparison of relative gray values among the three groups showed that COX-2 expression in cholesteoma epithelium was significantly higher than that of the other two groups,and the differences were statistically significant(p < 0.05).In the cholesteatoma group,the expression of cox-2 in the ossiculachain damaged group was higher than that in the non-ossiculachain damaged group,and the difference was statistically significant(p < 0.05).The relative gray values of PTEN in cholesteatoma group,granulation group and normal external auditory canal skin group were 0.4605±0.1449 、 0.7123±0.1283 and 0.8016±0.1197.Respectively compareing the relatively grey value to each other between the three groups,and to found that PTEN with the relative expression quantity minimum in cholesteatoma epithelium,compared with middle ear granulation and normal plane skin,the differences were statistically significant(p < 0.05).Compared middle ear granulation with normal external auditory canal skin,the differences were no statistically significant(p > 0.05).COX-2 and PTEN were correlated by Pearson assay,and there was a significant negative correlation between them in the relative expression level of cholesteoma epithelium(r=-0.659,p=0.00 < 0.05).2.Immunohistochemical results: COX-2 positive expression was localized in the cytoplasm,which was expressed to varying degrees in the middle ear cholesteoma,middle ear granulation and normal external auditory canal skin.COX-2 staining was the strongest in the middle ear cholesteatoma epithelium,showing a positive staining of medium-strength.Cells in all layers of the cholesteatoma epithelium tissue showed certain expression,among which the base membrane staining was the strongest(average optical density value: 0.361±0.126).COX-2 was weakly positive to moderately positive in granulation tissue(average optical density value: 0.262±0.072),mainly distributed in newborn capillaries and surrounding infiltrating inflammatory cells.The epithetlium of the normal external auditory canal was weakly positively expressed(average optical density value: 0.191±0.037).The expression of COX-2 in cholesteatoma group was significantly different from that in the middle ear granulation group and the normal external auditory canal skin group(p < 0.05).In the middle ear cholesteatoma group,the non-ossiculachain damaged group was mostly stained with moderate intensity(average optical density value: 0.251±0.024),while the ossiculachain damaged group was mostly stained with strong positive intensity(average optical density value: 0.397±0.124),and the difference was statistically significant(p < 0.05).The positive expression of PTEN protein was mainly located in the nucleus.In the middle ear cholesteatoma,PTEN showed weak positive staining(average optical density value: 0.257±0.082),mainly expressed in the epithelium of cholesteatoma.In the middle ear granulation,PTEN was moderate staining(average optical density value: 0.360±0.081),the positive cells mainly distributed in new capillaries and inflammatory cells.The skin in the normal plane,PTEN was the intensity of positive staining in(average optical density value: 0.416±0.065),in one of the layers of epithelial cells have certain expression.Comparison between the three groups of the average optical density value,the expression of PTEN in middle ear cholesteatoma is significantly lower than the middle ear granulation and normal plane skin,the difference was statistically significant(p < 0.05).In the middle ear cholesteatoma group,the expression level of PTEN in the ossiculachain destructed group(average optical density value: 0.229±0.065)is lower than the group without ossiculachain damaged(average optical density value: 0.342±0.072),the difference was statistically significant(p < 0.05).The expression of PTEN was found in the middle ear granulation tissue and the skin epithelium of the normal external auditory canal,and the difference was not statistically significant(p > 0.05).COX-2 and PTEN were correlated by Pearson assay,and there was a significant negative correlation between them in the relative expression level of cholesteoma epithelium(r=-0.659,p=0.00 < 0.05).Conclusion: 1.COX-2 protein expression was significantly increased in the middle ear cholesteatoma and chronic suppurative otitis media,suggesting that COX-2 may play an important role in the occurrence and development of cholesteatoma and chronic suppurative otitis media.2.PTEN protein expression was significantly decreased in middle ear cholesteoma,PTEN may induce the excessive proliferation and anti-apoptosis of cholesteoma epithelial cells through the PI3K-AKt signaling pathway,and participate in the occurrence and development of middle ear cholesteoma.3.In middle ear cholesteatoma group,the expression of COX-2 was significantly higher in the ossiculachain damaged group than in the non-ossiculachain damaged group,while the expression level of PTEN in the ossiculachain damaged group was significantly lower than that in the non-ossiculachain damaged group,suggesting that the high expression of COX-2 and the low expression of PTEN may also be involved in the invsaion destruction of middle ear cholesteatoma.4.The expression of COX-2 and PTEN in middle ear cholesteatoma showed a significant negative correlation,indicating that the low expression of PTEN and the high expression of COX-2 may have some kind of mutual regulation relationship,so that both of them are involved in the occurrence and development of middle ear cholesteatoma and the process of invsaion destruction. |
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