Expression And Significance Of Caspase-9 And Livin Associating With Apoptosis Proteins In The Middle Ear Cholesteatoma

Background and ObjectiveCholesteatoma is one of the most common chronic diseases of otology.The name called tumor stem from a cystic structure in the middle ear. The outer fibrous layer linked closely to the adjacent bones and tissues,the inner squamous layer is filled with shedding necrosis of epithelium, keratinized material and cholesterol crystals. It has tumor-like features of destruction, invasion, migration, differentiation variability and recurrence,all of which can lead to serious life-threatening intracranial complications.In recent years, scholars have researched the pathogenesis of cholesteatoma by testing means of immunization and molecular biology. They explore the mechanism of proliferation, apoptosis and bone destruction through the expression and distribution of local chemical media information molecule, signal transduction, substances of gene regulation and control. But its exact pathogenesis have not been fully clarified. At present,researchers think the apoptosis in the maligant tumor is restrained. However, there is still little reports about apoptosis of cholesteatoma, the results of which are not coincide with each other. There are two types of factors affecting cell apoptosis in human body,one type is pro-apoptotic factors such as RPR,Smac,H6Ra2,Caspases,the other is factors of inhibitor of apoptosis, for example Bcl-2,CrmA,P53,IAPs.As caspase family has the characteristics of self-activation and mutual activation. Apoptosis once is triggered,it will show cascade amplification. So the caspases cascade of protease solution is the core mechanism of apoptosis. Caspase-9 is the most important initiation factor of the caspases. IAPs is a new endogenous anti-apoptotic protein, and it is independent of Bcl-2. Livin dose not express or weakly express in most normal tissues, but a high expression in majority of tumor tissues. Livin plays a role in anti-apoptosis by combining caspase protein and inhibiting the caspase cascade activation reactions. Reseaches show that livin can bond directly Caspase-9 of unprocessed and fracture formation, which in turn restrains apoptosis.At present, most researchers prove that expression of pro-apoptotic factors is apparently high level in middle ear cholesteatoma, which also demonstrates that the ability of apoptosis is enhancement. But the results factors of apoptosis inhibitor are not agree with cholesteatoma in the middle ear. We hypothize that Caspase-9 combined with livin participate in the process of cholesteatoma pathophysiology, what's the relationship of them? This study is to explore the levels of expression and the correlation between Caspase-9 of apoptosis protein and apoptosis protein of livin inhibitor in the middle ear cholesteatoma, demonstrating the roles of Caspase-9 protein and livin protein in the development and progession of the middle ear cholesteatoma.At last,we disclose the pathogenesis and offer new ideas for the clinical treatment of middle ear cholesteatoma.Methods1. resource and tissues handling:All samples were obtained from patients who underwent tympanomastoid surgery for middle ear cholesteatoma in the First Affiliated Hospital of Zhengzhou university at the fist time. All specimens were fixed in 10% neutral formaldehyde solution, and then embedded in paraffin. Cuts were prepared.2. Slides were deparaffinized and dehydrated in graded alchol. The deparaffinized sections were soaked in 0.01-mol/L citrate buffer and heated in a microwave oven for 20 minutes, followed by blocking of endogenous peroxidase with 0.3% hydrogen peroxidase in methanol. Nonspecific binding was blocked with 10% normal goat serum for 30 minutes.After a brief rinse, the sections were immunoreacted with a caspase-9 polyclonal antibody(livin polyclonal antibody) at room temperature for 30 mintues, the sections were washed in phosphate-buffered saline. After treatment with avidin-biotin complex for 30 minutes, a solution of 3,3'-diaminobenzidine was then applied as a chromogen. The sections were counterstained with hematoxylin, and the specimens were mounted with coverslips using mounting medium, the immunohistochemical staining results were analysized quantitatively by the Olympus BH-2 optical microscope and the microscopic image analysis system of Biosens Digatal Imaging Systerm.Results1.①Caspase-9 was stained in the cytoplasm and membrane, found as a brown-yellow color in the all layers of cholesteatoma epithelium,but positive cells located mainly in granula and spinous layers of cholesteatoma. In normal skin of exteral ear canal, caspase-9 was detected in the granular and spinous layer, too.②Livin was stained in the cytoplasm but not in the nucleus,it was localized throughout all the layers of epithelium. In normal skin of the external ear canal, livin was distribued in the basal and suprabasal layers.2.①The average gray values and positive cells of caspase-9 were 124.68±67.23 and 87.21±9.45 in the middle ear cholesteatoma,68.25±47.17 and 35.13±10.17 in the normal skin of the external ear canal. the expressions of caspase-9 were signifincantly higher in the middle ear cholesteatoma and the difference was statistically significant.②The average gray values of livin was 75.66±56.54 in the middle ear cholesteatoma but 56.99±49.07 in the normal skin of exteral ear canal. The number of positive cells was 38.17±10.21 and 19.56±4.96.The expressions of livin in the middle ear cholesteatoma were signifincantly higher than those in normal skin of the external ear canal,the difference was statistically significant,③both Caspase-9 and livin protein had a positive staining in 14 cases and negative staining in 4 cases, livin had a positive staining but absence of Caspase-9 staining in 1 case, Caspase-9 had a positive staining but livin had a negative staining in 4 case of the Correlation analysis. Their expressions in the middle ear cholesteatoma were statistically significant by the statistical analysis of fourfold method of X2 test,p<0.05.3. Conclusion1. The expression of caspase-9 was significantly higher in the middle ear cholesteatoma,which demonstrates the ability of apoptosis is intensive in the middle ear cholesteatoma.2. The expression of livin in the middle ear cholesteatoma was signifincantly stronger than those in normal skin of the external ear canal, the test of which displayed that the capacity of restraining the apoptosis also enhancement.3. The expression of caspase-9 protein and livin protein in the middle ear cholesteatoma made the apoptosis in the middle ear cholesteatoma reach a new balance,which was different from maligant tumor.