蛋白激酶C激活的荧光共振能量转移分析  

Fluorescence resonance energy transfer analysis for detecting protein kinase C activation

作　　者：李俊青[1] 江建明[1] 冯瑞强[1] 蒋晖[1] 闫文娟[2] 杨德鸿[1] 

机构地区：[1]南方医科大学南方医院脊柱骨科,广东广州510515 [2]南方医科大学南方医院口腔科,广东广州510515

出　　处：《南方医科大学学报》2011年第11期1867-1870,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30973061);广东省自然科学基金项目(9151051501000087)~~

摘　　要：目的当前,检测蛋白激酶C(PKC)激活的方法尚缺乏灵敏性或直接性,这里我们提供了一种新的直接而灵敏的方法——利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术来构建检测PKC激活的方法。方法将表达PKC激活的报告分子(CKAR)的质粒单独转染或与表达甲状旁腺素1型受体(PTHR1)的质粒共转染HEK293细胞,培养72 h后用激光共聚焦显微镜检测FRET的变化,并以此判断甲状旁腺素或佛波酯是否激活PKC。结果在只转染CKAR质粒的HEK293细胞,佛波酯降低了CKAR分子的FRET效率,并使青色荧光与黄色荧光的比值(C/Y)增加,而PTH(1-34)未能改变C/Y的值。在共转染了CKAR和PTHR1质粒的HEK293细胞,PTH(1-34)则使C/Y增加。结论PKC激活报告分子可用于检测PKC的激活,该方法也可作为PKC相关信号转导的实验平台。Objective To establish a sensitive and direct method for detecting the activation of protein kinase C（PKC） using fluorescence resonance energy transfer（FRET） technique.Methods HEK293 cells were transfected with C kinase activity reporter（CKAR） plasmid or/and parathyroid receptor 1 plasmid,and after incubation for 72 h,the fluorescence resonance energy transfer was measured with or without parathyroid or TPA stimulation.Results TPA reduced the efficiency of FRET and increased the emission ratio of CFP/YFP（C/Y） in HEK293 cells transfected with CKAR.PTH（1-34） could increase the emission ratio of C/Y in HEK293 cells co-transfected with CKAR and PTHR1 but not in cells transfected with CKAR.Conclusion FRET analysis using CKAR can be utilized to detect the activation of PKC,which provides a useful means for studying the signaling pathways associated with PKC.

关 键 词：蛋白激酶C 荧光共振能量转移 PKC激活报告分子 

分 类 号：R341[医药卫生—基础医学]