5-HT_(1A)受体蛋白在PC12细胞膜转运的动态变化  

Visualization of serotonin 1A receptor trafficking in neuron-like PC12 cells

作　　者：金艳燕[1] 吕琼[1] 闫竹青[1] 高尔静[1] 赵春礼[1] 张进禄[1] 徐志卿[1] 

机构地区：[1]首都医科大学神经生物学系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,北京100069

出　　处：《基础医学与临床》2010年第5期487-491,共5页Basic and Clinical Medicine

基　　金：国家重点基础研究发展计划973(2007CB512307);北京市自然科学基金重点项目(09G0013);人事部留学人员科技活动择优资助项目

摘　　要：目的在神经元样PC12活细胞上进行实时、可视和定量研究5-羟色胺1A受体的时空分布、膜转运和信号传导机制。方法运用RT-PCR方法获得小鼠5-HT1A基因,并插入到pEGFP-N1真核表达载体中。采用阳离子脂质体方法将质粒转染至PC12细胞和HEK293细胞中,通过G418筛选出稳定表达5-HT1A-EGFP的PC12细胞系。运用激光共聚焦成像系统观察活细胞中5-HT1A-EGFP的表达情况,利用光漂白荧光恢复(FRAP)技术在PC12细胞膜局部漂白后观察5-HT1A-EGFP荧光蛋白在细胞膜上转运的情况。结果克隆所获得的小鼠5-HT1A基因是准确的。5-HT1A-EGFP蛋白清晰的分布于PC12和HKE293细胞膜上。通过FRAP技术观察到漂白区域的细胞膜在100s内有部分恢复,说明受体在细胞膜上发生转运。结论建立了稳定表达5-HT1A-EGFP融合蛋白的PC12细胞系,并利用活细胞成像和FRAP技术观察分析并证实了5-HT1A受体在PC12细胞的膜表面的表达和转运的动态变化。Objective To explore the mechanisms of trafficking and signaling of serotonin 1A receptor（5-HT1A）and its spatiotemporal distribution in living cells.Methods The mouse 5-HT1A gene amplified by RT-PCR was recombined into pEGFP-N1 vector and the EGFP coding sequence was located in-frame at the C-terminal end of the 5-HT1A receptor.The 5-HT1A-EGFP was transfected into neuron-like PC12 cells as well as HEK293.The transfected cells were visualized using confocal microscopy,the mobility of 5-HT1A-EGFP was monitored by live measurements and fluorescence recovery after photobleaching.Results The 5-HT1A gene was identitical with the published gene sequence NM_008308.4 and a 5-HT1A-EGFP fusion construct was created.After stable transfection of the plasimd into a PC12 cell line and analysis with a confocal laser scanning microscopy,the EGFP-tagged 5-HT1A was predominantly associated with the plasma membrane,but some intracellular vesicles in the perinuclear region alsocontained the fusion protein.The predominant localization of 5-HT1A-EGFP at the plasma membrane was confirmed in transiently transfected HEK293 cells.Bleached fluorescence was partialy recovered in 100 seconds,indicating that the 5-HT1A-EGFP was mobiled on the membrane.Conclusion Spatiotemporal distribution and mobility of 5-HT1A tagged with EGFP can be monitored in the 5-HT1A-EGFP stable PC12 cell line,which could be an excellent neuron-like experimental cell model for research of 5-HT1A trafficking and signaling.

关 键 词：5-羟色胺1A受体(5-HT1A) G蛋白偶联受体(GPCRs) 绿色荧光蛋白(GFP) PC12细胞 光漂白荧光恢复技术(FRAP) 

分 类 号：R73-3[医药卫生—肿瘤]