用一种新方法检测尼古丁减轻鱼藤酮对UPS的毒性  

A new method to detect nicotine's effect in alleviating rotenone-induced toxicity of ubiquitin-proteasome system

作　　者：石婕[1] 邓永宁[1] 刘洁[1] 屈秋民[1] 

机构地区：[1]西安交通大学医学院第一附属医院神经内科,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2014年第1期132-135,138,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

摘　　要：目的建立一种动态检测泛素-蛋白酶系统(UPS)活性的新方法,并利用此方法观察尼古丁对鱼藤酮诱导的UPS功能障碍的影响。方法合成UPS降解信号CL1,将其与含有绿色荧光蛋白的质粒pEGFP-C1连接,构建CL1-pEGFP-C1质粒并转染PC12细胞,经G418筛选后得到稳定表达的CL1-pEGFP-C1细胞系。蛋白酶抑制剂MG132、鱼藤酮或尼古丁+鱼藤酮处理细胞,应用免疫印迹技术检测细胞内GFP含量的变化,荧光显微镜观察细胞荧光强度的变化。结果稳定转染的CL1-pEGFP-C1细胞无绿色荧光,而MG132处理后细胞内绿色荧光显著增加,且随MG132浓度增加荧光强度逐渐增加,GFP表达逐渐增高(P<0.05)。鱼藤酮处理后细胞内绿色荧光显著增加,GFP含量明显高于非处理组(P<0.05)。低浓度尼古丁处理组细胞内荧光强度显著降低,GFP蛋白表达显著减少,高浓度尼古丁处理组细胞内荧光强度降低更加显著,GFP蛋白表达减少更明显(P<0.05)。结论成功建立了能够在活细胞中全面、动态检测UPS活性的方法。鱼藤酮可降低UPS活性,尼古丁可减轻鱼藤酮引起的UPS功能障碍。Objective To establish a new method for dynamically detecting the activity of ubiquitin- proteasome system （UPS） in living cells and observe the influence of nicotine on rotenone-induced dysfunction of UPS. Methods By adding a sequence coding CL1, a degradation signal for UPS, the plasmids pEGFP-C1 expressing GFP were reconstructed into CLI-pEGFP-C1 expressing GFP. PC12 cells were transfected with CL1- pEGFP-C1, and then the stable cell lines expressing CLI-pEGFP-C1 were obtained by G418 selection. After treatment with the proteasome inhibitor MG132, rotenone and nicotine -- rotenone, the level of GFP in CL1- pEGFP-C1 stable cell line was analyzed by immunoblotting and the fluorescent intensity of GFP was examined under fluorescent microscope. Results It was found that MG132 and rotenone treatment obviously elevated the level and fluorescent intensity of GFP in CLI-pEGFP-C1 stable cell line in a dose-dependent manner （P 〈 0.05）. Nicotine could block the toxic effect of rotenone on UPS partially by alleviating the level and fluorescent intensity of GFP. Oonclusion A new method for dynamically detecting the activity of UPS in living cells has been successfully established. Rotenone can reduce the activity of UPS and nicotine can alleviate rotenone-induced dysfunction of UPS.

关 键 词：泛素-蛋白酶系统 尼古丁 鱼藤酮 帕金森病 

分 类 号：R742.5[医药卫生—神经病学与精神病学]