钙调素在生长相关蛋白-43调控细胞周期中的作用  

Role of growth associated protein-43 on regulating the cell cycle via calmodulin

作　　者：程蓓[1] 赵君朋[1] 徐群渊[1] 

机构地区：[1]首都医科大学北京神经科学研究所,北京市神经再生修复研究重点实验室,教育部神经变性病重点实验室,北京100069

出　　处：《解剖学报》2012年第3期340-346,共7页Acta Anatomica Sinica

基　　金：国家重点基础研究发展计划资助项目(2007CB947704)

摘　　要：目的探讨钙调素-43(CaM)在生长相关蛋白(-43)(GAP-43)调控细胞周期过程中的作用。方法利用反转录病毒体系构建表达不同活性状态的GAP-43(即野生型、非磷酸化型和假磷酸化型GAP-43)NIH3T3细胞模型,并分别命名为表达野生型GAP-43(3T3-G)、表达非磷酸化GAP-43(3T3-A)和表达假磷酸化GAP-43(3T3-D);另设对照组(转染空载体NIH3T3细胞)。通过免疫组织化学及Western blotting鉴定各型细胞模型转入GAP-43的情况;应用免疫共沉淀检测各型GAP-43与CaM的功能关系;应用细胞生长曲线、BrdU累积标记法、BrdU脉冲标记法测定各型转GAP-43细胞的细胞周期。结果免疫组织化学和Western blotting结果显示,NIH3T3细胞能够表达转入的各型GAP-43。其中,3T3-A细胞增殖速度较其他细胞减缓。免疫共沉淀显示,3T3-A中有GAP-43和CaM相互作用表现;3T3-G中GAP-43和CaM有少量相互作用;3T3-D与3T3-P中则相互作用。累积BrdU测定,脉冲BrdU测定M期的实验表明,3T3-P细胞无明显周期改变;3T3-D与3T3-G细胞的周期延长;3T3-A细胞的周期明显延长并显示G1期明显延长。结论表达GAP-43的细胞其增殖表现可能是由于GAP-43结合了CaM,使之与Ca2+的结合减少而引起细胞周期(尤其是G1期)的延长。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 Gl phase. 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 Ca2＋ , causing an extension of cell cycle, especially for the phase of G1.

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 

分 类 号：R742.5[医药卫生—神经病学与精神病学]