Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology · 2017
The Role of Rho/Rho-Kinase Pathway in the Pathogenesis of Cholesteatoma.
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Yeşilova Mesut, Görür Kemal, Ismi Onur, Özcan Cengiz, Büyükafşar Kansu
Аннотация
OBJECTIVE: To assess the role of Rho/Rho-kinase pathway in the pathogenesis of cholesteatoma.
MATERIALS AND METHODS: Thirty-eight patients with cholesteatoma, who had gone mastoidectomies were enrolled in this prospective study. Cholesteatomas matrix (CM) and a piece of the external ear canal skin (EECS as control) were taken and transferred to the liquid nitrogen and kept at -86 °C for Rho A and Rho-kinase (ROCK) analysis with Western blotting and commercial ELISA kits (Cell Biolabs Inc., San Diego, CA). The tissues were homogenized by an appropriate ice-cold lysis buffer. Following centrifugation, the supernatant was taken and total protein amount was detected by the Bradford method. Thereafter, tissue homogenates were subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis electrophoresis then transferred to nitrocellulose membrane where it was treated with specific monoclonal primary antibody against to ROCK-2 and HRP-conjugated seconder antibody, respectively. The protein blots were visualized with commercial x-ray film and dansitometrically analyzed by the Scion Image Program (Cell Biolabs Inc., San Diego, CA). In another series of experiments, Rho-kinase activities were assessed by ROCK-2 ELISA kits.
RESULTS: There were no statistical differences in Rho A translocation between CM and EECS. However, ROCK activity was found to be lower in CM than EECS as detected by ELISA kits. Furthermore, ROCK protein expression was also significantly lower in CM than EECS as demonstrated by Western blotting.
CONCLUSION: Given Rho-kinase could take essential roles in cell differentiation, the results of this study implicate that down-regulated Rho-kinase could be responsible for the keratinocyte undifferentiation seen in cholesteatoma pathogenesis.
MATERIALS AND METHODS: Thirty-eight patients with cholesteatoma, who had gone mastoidectomies were enrolled in this prospective study. Cholesteatomas matrix (CM) and a piece of the external ear canal skin (EECS as control) were taken and transferred to the liquid nitrogen and kept at -86 °C for Rho A and Rho-kinase (ROCK) analysis with Western blotting and commercial ELISA kits (Cell Biolabs Inc., San Diego, CA). The tissues were homogenized by an appropriate ice-cold lysis buffer. Following centrifugation, the supernatant was taken and total protein amount was detected by the Bradford method. Thereafter, tissue homogenates were subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis electrophoresis then transferred to nitrocellulose membrane where it was treated with specific monoclonal primary antibody against to ROCK-2 and HRP-conjugated seconder antibody, respectively. The protein blots were visualized with commercial x-ray film and dansitometrically analyzed by the Scion Image Program (Cell Biolabs Inc., San Diego, CA). In another series of experiments, Rho-kinase activities were assessed by ROCK-2 ELISA kits.
RESULTS: There were no statistical differences in Rho A translocation between CM and EECS. However, ROCK activity was found to be lower in CM than EECS as detected by ELISA kits. Furthermore, ROCK protein expression was also significantly lower in CM than EECS as demonstrated by Western blotting.
CONCLUSION: Given Rho-kinase could take essential roles in cell differentiation, the results of this study implicate that down-regulated Rho-kinase could be responsible for the keratinocyte undifferentiation seen in cholesteatoma pathogenesis.