| Objective: In this experiment,using the fact that cholesteatoma is similar to the tumor’s adhesion and the basic condition of tumor invasion is adhesion and matrix degradation as a entry point,the expression of CD44V6 and MMP-9 were detected in middle ear cholesteatoma to explore their role in the invasion of cholesteatoma and their correlation.Methods:1.Immunohistochemistry: The Paraffin blocks of patients who underwent Modified mastoidectomy and tympanoplasty for middle ear cholesteatoma or chronic suppurative otitis media with complete medical records were collected from May 2013 to August 2016 in Department of Otolaryngology of the First Affiliated Hospital of Southwest Medical University.The experimental group was divided into two groups: cholesteatoma group and granulation group,Among them,there were 22 cases of cholesteatoma group,and there were a total of 11 cases in granulation group.The control group consisted of 10 specimens of normal external auditory canal epithelium which were randomly obtained from the patients with chronic suppurative otitis media during the surgery from August 2016 to December 2016.All specimens were confirmed by pathology.We used Immunohistochemical method and Image Pro Plus image analysis software to detect the expression of MMP-9 and CD44v6 in 22 cases of middle ear cholesteatoma,11 cases of granulation tissue and 10 cases of normal external auditory canal.and used spss statistical software for datastatistics.The expression of three groups was compared and the correlation between the two molecules and the destruction of cholesteatoma and the correlation of the two proteins were analyzed.2.RT-PCR: The intraoperative specimens of the Patients who underwent Modified mastoidectomy and tympanoplasty for middle ear cholesteatoma or chronic suppurative otitis media with complete medical records were be collected From August to December2016 in Department of Otolaryngology of the First Affiliated Hospital of Southwest Medical University.The experimental group was divided into two groups: cholesteatoma group and granulation group,Among them,there were 10 cases of cholesteatoma group,and there were a total of 10 cases in granulation group.The control group consisted of 10 specimens of normal external auditory canal epithelium which were randomly obtained.All specimens were confirmed by pathology.Then,the specimens were directly immersed in RNAwait(non frozen tissue RNA preservation solution)and were palced in the refrigerator at4 degrees centigrade overnight.The second day,we moved it to the refrigerator at-20 degrees centigrade for RT-PCR.We Designed the upstream and downstream primers of CD44v6 and matrix metalloproteinase 9.The expression of CD44v6 and matrix metalloproteinase 9 mRNA was detected by RT-PCR method,and the results were compared and analyzed.Results: 1.Immunohistochemistry:The positive expression of CD44V6 was located on the cell membrane,and was expressed in different degrees in the middle ear cholesteatoma epithelium,granulation,and external auditory canalepithelium.In the middle ear cholesteatoma epithelium,the staining intensity was the strongest,and the intensity of expression is medium-strong positive.The expression of the basement membrane of the epithelium was the strongest..(Average optical density0.2170±0.1023).The epitheliums of the external auditory canal showed weakly positive to moderately positive(Average optical density0.1025±0.0204),In granulation tissue,the expression of CD44V6 protein was weakly positive(Average optical density 0.0095±0.0083).There was significant difference between the cholesteatoma group and the external auditory canal skin group(p<0.05).In no obvious destruction of ossicles group,the expression of CD44V6 protein showed moderate staining(the average optical density 0.1523± 0.0343),and In ossicle destruction group,the expression of CD44V6 protein showed strong positive(the average optical density 0.2811±0.0109),the difference was statistically significant(p<0.05).The positive expression of MMP-9 was located in the cytoplasm of the cells.In cholesteatoma specimens,MMP-9 protein did not express in epithelium,mainly expressed in the subepithelial connective tissue and inflammatory cells such as neutrophils and plasma cells,and the expression was mainly diffuse distribution,and a few were distributed(0.0250±0.0128);In granulation tissue,the expression of MMP-9 protein was mainly expressed in the newborn capillaries and the inflammatory cells distributed in the periphery(0.0127±0.0921).In the skin tissue of the external auditory canal,It was mainly expressed in a few inflammatory cells in the subepithelial,and showed weak positive expression(0.0046±0.0015).We compared three data bases by multiple comparison,the differences were statistically significant(p<0.05).In no obvious destruction of ossicles group,the average optical density was 0.0130±0.0031,and In ossicle destruction group,the average optical density was 0.0320±0.0107,the difference was statistically significant(p<0.05).The average optical density of CD44V6 and MMP-9 expression was significantly correlated with Pearson(r=0.525,p=0.017<0.05).Conclusions:1.MMP-9 is expressed increasely in middle ear cholesteatoma and the new capillaries and inflammatory cells of granulation tissue which caused invasion and destruction of cholesteatoma through regulating angiogenesis,enhancing the nutritional support of cholesteatoma and promoting the degradation of extracellular matrix;2.CD44V6 is expressed in cholesteatoma epithelium which promoted the occurrence and development of cholesteatoma by up regulating inflammatory related genes;3.The expression of CD44V6 and MMP-9 in the ossicle destruction group of cholesteatoma was increased,suggesting that both of them may play an important role in the invasion and destruction of cholesteatoma;4.In cholesteatoma tissues,the expression of CD44V6 enhanced the anchoring effect of MMP-9 and cholesteatoma tissue,and then promote the degradation of extracellular matrix;and may be acting on the HA/CD44 signaling pathway,the increased expression of MMP-9 and MMP-9 synergistically promote the invasiveness of cholesteatoma damage. |
