关键词: 生长相关蛋白-43, NIH3T3细胞, 钙调素, 细胞周期, BrdU, 免疫印迹法, 免疫组织化学

Abstract: Objective To explore the role of growth association protein-43 (GAP-43) in regulation of cell cycle via the interaction with calmodulin (CaM).Methods A series of NIH3T3 cell models, which expressed different types of GAP-43, i.e., 3T3-G (expression of wild-type GAP-43), 3T3-A (expression of non-phosphorylated GAP-43), and 3T3-D (expression of phosphorylated GAP-43), were established by using a retroviral expression system. A separate group of NIH3T3 cells, which was transfected only with an empty vector, were used as a control. Immunofluorescence and Western blotting were used for detecting expression of individual protein in transfected NIH3T3 cells and the co-immunoprecipitation was applied to detect the interaction of different types of GAP-43 with CaM. The cell counting, cumulative BrdU labelling, and the percentage of labeled mitotic figures (PLM labeling) were introduced to determine the cell cycle in different cell models.Results The cell models expressing different types of GAP-43 were successfully created and confirmed by immunocytochemistry and Western blotting. The co-immunoprecipitation showed a clear interaction between GAP-43 and CaM in 3T3-A cells, a little in 3T3-G cells, and no such interaction in either 3T3-D or 3T3-P cells. No significant change of cell cycle was observed by cumulative BrdU measurement,pulse BrdU test and M phase test in 3T3-P cells, while certain extensions were seen in 3T3-D and 3T3-G cells. In 3T3-A, the cell cycle was shown to be prolonged with an extension of G1phase.Conclusion The proliferation of NIH3T3 cells, caused by GAP-43 expression may be due to an interaction between GAP-43 and CaM, which may reduce binding capacity of CaM to the Ca SUP>2 +/SUP>, causing an extension of cell cycle, especially for

Key words: GAP-43, NIH3T3 cells, Calmodulin, Cell cycle, BrdU, Western blotting, Immunohistochemistry