| Background The middle ear cholesteatoma is a common disease I of Otolarynology and characterized by hyperproliferative keratinated squamous epithelium in the middle ear cavity. Cholesteatoma epithelium cells are not only hyperprolifetative but also overapoptosis. But The mechanism of middle ear clesteatoma is not clear yet. In molecular biology, the domestic and international researchers have worked hard to study the mechanism of cholesteatoma recently. Many experiments confirmed that apoptosis is controlled by its relative genes. And many diseases do something with cell apoptosis and the turbulence of its controlled genes. TGF-β1 and p21WAF1 are two relative factor of apoptosis. TGF-β1 restrained cell proliferation with cyclin, start with cell apoptosis and accelerated the period of cholesteatoma epithelium apoptosis. It also can stimulate the expression of other genes to adjust cell apoptosis, such as p53,p27,p21. As the same time TGF-β1 can affect the bone and connective tissues, and correlated with inflammation. p21WAF1 is a prevenient controlling factor of p53 gene, and a cyclin-dependent kinase-inhibitor to repress most cyclin-dependent kinases. p21WAF1 depended on p53 to band cyclin-CDK and make CDK lose activity, blocking CDK to phosphate pRB. The cell stage stopped in G1. p21WAF1 participated in the period of cell apoptosis,DNA replication, growth, dividing, decrepituding etc. TGF-β1 can induce the expression of p21WAF1 to accelerate cell apoptosis of cholesteatoma epithelium. Objective Study the expression and correlation of TGF-β1 and p21WAF1 in middle ear cholesteatoma and Discuse their effect in cholesteatoma. Methods 20 samples of middle ear cholesteatoma we studied came form the patients who were operated in our hospital during Mach.2004 to Jun.2005, and all of them had been verified to be cholesteatoma by pathology. 10 samples were normal skin of the external ear canal acquired from tympanoplasty at the same time. The wax specimens were sliced into 4μm sections continuously. We used the immumohistochemical technology with streptavidin-peroxidase TM. The primary antibodies were mouse anti-human P21WAF1monocional antibody and rabbit anti-human TGF-β1 polyclonal antibody separately, and they were both instant antibodies. PBS was used for negative control instead of the primary antibody. The main steps of operation: After deparaffinization and washing, we used saline sodium citrate solution for antigen reparetion. Edogenous peroxidase of the sections were inhibited with a methanolic solution and then blocked with normal barbary serum to reduce background. Hence we fixed primary antibody (4℃,vernight), postfixed IgG labeled by biotin, and color development was performed with DAB, The sections afterstained and occluded by neutral balsam. Under microscopic observation at magnification×400, we selected five random visual fields and calculated the positive cells, then with the formula LI=∑n/∑N×100% we get labeling index(LI). We dealed with the data with SAS system software, used x2 test and the paired samples Spearman-test to compare the meansof paired variables with a level of significance of p<0.05. Results: ①.The result showed that p21WAF1 and TGF-β1 could be detected in middle ear cholesteatoma and in normal ear cananl shin. p21WAF1 was located at karyon, taking on yellow-brown pellet; TGF-β1 was located at cytoplasmic, taking on yellow-brown pellet. ②13 cases of p21WAF1 had positive cells in 20 cholesteatoma samples, positive rate was 65%; There almost no positive cells in normal ear canal skin. And p21WAF1 positive cells were detected in each layers, whose intensity was high to weak, and its LI was 28.9%±6.7%. Compared with normal ear canal skin, the expression of p21WAF1 increased in middle ear cholesteatoma epithelium(P<0.05). ③TGF-β1 positive rate was 90% in 20 middle cholesteatoma samples, whose intensity was high to weak, and 50% in normal ear canal skin. The positive cells mostly in spine layer and in granular layer, low intensive, light-brown, and its LI was 13.3%±4.9%; In middle ear cholsteatoma epithelium, positive cells scattered among all layers of the epithelium, and stained strangest in spine layer, high intensive, strong-brown, and its LI was 32.3%±5.7%. They had obvious difference ( P<0.05 ) . ④In cholesteatoma epithelium samples, p21WAF1-LI was 28.9%±6.7%, and TGF-β1-LI was 32.3%±5.7%. They didn't have significant difference by statistics. But in cholesteatomatous tissues they were correlated closely by Spermen test. Conclusion ①The result showed the expression of TGF-β1 and p21WAF1 increase in middle ear cholesteatoma epithelium, which confirmed that TGF-β1 and...
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