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대한Helicobacter 연구학회지 2002;2(2):148-155.
Published online November 21, 2002.
Immunofluorescence Study of Rho GTPases in Cultured AGS Cells Infected with Helicobacter pylori
Helicobacter pylori로 감염된 AGS 세포에서 면역형광반응을 이용한 Rho-GTPase의 발현
허병원·전훈재·정록선·김용식·김영선·박철희·진윤태·이홍식·엄순호·이상우·최재현·김창덕·류호상·박은경*·민혜정*·엄창섭*현진해
고려대학교 의과대학 내과학교실, 소화기 연구소, *유전병 연구소
Abstract

Background/Aims: It has been suggested that the adhesion of H. pylori on the gastric epithelial cells are mediated by Bab A adhesin, Lewis antigen. However, precise mechanism of adhesion has not been elucidated yet. In this study, expression of different members of Rho-GTPases are investigated in the cultured AGS cells exposed to H. pylori by immunofluorescent staining and western blot analysis. Materials and Methods: AGS cell line was purchased from ATCC and cultured in DMEM with 10% FBS. Cultured cells were exposed to H. pylori at a density of O.D. of 1 at 600 nm for 1, 2 and 4 hrs. The cells were fixed in 4% paraformaldehyde. Fixed cells were washed and processed for immunofluorescence labeling. Primary antibodies against Rho, Rac1, and Cdc42 were labeled and visualized with FITC-labelled secondary antibody. Florescent labelled cells were observed with Zeiss Axioskop fluorescent microscope with appropriate filter sets. Proteins from the same cultured cells were extracted and examined by Western blot analysis. Results: Immunofluorescent labeling for Rho appeared as fine dots, which increased in size with the progress of adhesion. At 4 hr Rho-immunoreactivity was concentrated at the center of AGS cells. Rac1-immunoreactivity was present at the periphery and formed a reticulum on the top surface of AGS cells. Immunoreactivity for Cdc42 was not prominent. Western blot analysis confirmed the immunofluorescent labeling. Conclusions: These results showed the changes of different Rho-GTPases by H. pylori adhesion, and suggested that Rac1 was most important in the early interaction of H. pylori to the host cells. The exact mechanism for Rho-GTPases changes and the roles of different rho-GTPases in H. pylori pathophysiology need to be further investigated. (Korean J Helicobacter Res Prac 2002;2:148-155)

Key Words: AGS cells, Helicobacter pylori, Immunofluorescene, Rho, Rho- GTPase
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