Role Of Phosphatidylinositol3-kinase/Protein Kinase B(PI3K/PKB)Signal Transduction Pathway In The Pathogenesis Of Middle Ear Cholesteatoma

Objective:To investigate the expression and significance of P-EGFR, P-Akt, cyclinD1and PCNA in middle ear cholesteatoma.Method:P-EGFR, P-Akt, cyclinD1and PCNA expression were detected by immunohistochemical method in40cholesteatoma samples and20samples of normal external auditory canal (EAC) epithelium.Results:The positive rate of P-EGFR in cholesteatoma tissues was65.0%(26/40) compared to20.0%(4/20) in normal EAC epithelium; the percentage of P-Akt expression in the cholesteatoma tissues was72.5%(29/40) and that in normal EAC epithelium was35.0%(7/20); the positive rate of cyclinD1in cholesteatoma tissues was62.5%(25/40) compared to10.0%(2/20) in normal EAC epithelium; the percentage of PCNA expression in the cholesteatoma epithelium was80.0%(32/40) and that in normal EAC epithelium was45.0%(9/20). Protein expressions of P-EGFR, P-Akt, cyclinD1and PCNA in cholesteatoma epithelium were significantly increased when compared with normal EAC epithelium (P<0.05). In cholesteatoma, a positive correlation was found between P-EGFR and P-Akt; a positive correlation was also found between P-Akt and cyclinD1, cyclinD1and PCNA, respectively.Conclusion:1. Protein expressions of P-EGFR, P-Akt, cyclinD1, and PCNA in cholesteatoma epithelium were significantly increased when compared with normal EAC epithelium. In cholesteatoma, a positive correlation was established between P-EGFR and P-Akt, P-Akt and cyclinD1, cyclinD1and PCNA, respectively. These results showed that EGFR may be activated with the up-regulation of P-Akt, cyclinD1, and PCNA in cholesteatoma epithelium.2. The activation of EGFR/PI3K/Akt signaling transduction pathway may be involved in the cellular hyperplasia mechanism in cholesteatoma epithelium. Objective:To establish a method for primary culture and identi-fication of human external ear keratinocytes and to investigate its morph-ologic and biological features so as to provide sufficient seeding cells for the study in part III.Method:Skin tissue of external ear canal was collected in middle ear surgery. The primary human external ear keratinocytes were cultured with tissue explants method. The keratinocytes were isolated from skin by selected dissociation. The cultured cells were identified by morphology and immunofluorescence. The biological features were studied under inverted phase contrast microscope and growth curves were plotted.Results:5days later, we can see cell colonies made up of a few cells, and the cells gradually grew outwards from the colonies.15days later, the cultured cells grew into monolayer, they were polygonal, and showed "cobblestone-like" appearance. The cells had a positive reaction to antibodies against keratin. The morphological and biological characteri-stics were similar to those of typical keratinocytes.Conclusion:The cell line established in this study was proved to be primary human external ear keratinocytes. It has similar morphological and biological characteristics with typical skin keratinocytes. It may be a suitable model for in-depth study on the molecular mechanisms of middle ear cholesteatoma. Objective:To investigate the impact of EGF on the proliferation and migration in human external ear canal keratinocytes and to examine the protein expressions of EGFR, P-EGFR, Akt, P-Akt and cyclinD1in external ear canal keratinocytes stimulated with or without EGF.Method:Human external ear canal keratinocytes were stimulated with EGF in the presence or absence of AG1478or Wortmannin for the indicated periods of time. Then, cell proliferation was measured by MTT assay; cell cycle and cell motility were analysed by Flow Cytometer and Transwell migration assay; and protein expressions of EGFR, P-EGFR, Akt, P-Akt and cyclinD1were examined by Western blotting.Results:1. MTT assay:EGF contributed to proliferation of external ear canal keratinocytes as the extension of stimulating concentration and time; however, AG1478and Wortmannin potently inhibited EGF-stimulated proliferation of external ear canal keratinocytes.2. Flow Cytometer:Human external ear canal keratinocytes stimulated with EGF demonstrated the decreases in phase G1and increases in phase S, while pretreatment with AG1478or Wortmannin before the addition of EGF resulted in the increases in phase G1and decreases in phase S.3. Western blotting:We stimulated human external ear canal keratinocytes with EGF in the presence or absence of AG1478or Wortmannin for the indicated periods of time. EGF mediated a rapid and robust phosphorylation of EGFR and Akt in the cells, followed by a significant up-regulation of cyclinD124h later. AG1478and Wortmannin potently inhibited EGF-stimulated up-regulation of P-EGFR, P-Akt, and cyclinD1in external ear canal keratinocytes. The total amount of EGFR and Akt were constant.4. Transwell migration assay:EGF promoted the motility of external ear canal keratinocytes. After24h, cells on the bottom of the membranes increased significantly (P<0.05). AG1478and Wortmannin potently inhibited EGF-stimulated cell motility (P<0.05).Conclusion:1. In vitro, EGF stimulated the growth of cultured human external ear canal keratinocytes via the activation of EGFR/PI3K/Akt signaling transduction pathway, while AG1478and Wortmannin potently inhibited EGF-stimulated growth of human external ear canal keratinocytes.2. In vitro, EGF promoted the motility of human external ear canal keratinocytes via the activation of EGFR/PI3K/Akt signaling transduction pathway, while AG1478and Wortmannin potently inhibited EGF-stimulated cell motility.3. The activation of EGFR/PI3K/Akt signaling transduction pathway may be involved in the etiopathogenesis of middle ear cholesteatoma.