AP-1蛋白c-Jun与Fral形成二聚体促进类多巴能神经细胞SH-SY5Y存活  

A dimer formed by activator protein-1 family proteins c-Jun and Fral increases viability of dopaminergic neurons SH-SY5Y

作　　者：何伟文[1] 何国振[1] 伍建伟[1] 梁建峰[1] 袁忠民[1] 

机构地区：[1]广州医科大学附属第二医院神经外科、广州医科大学神经科学研究所广东省重点实验室、神经遗传与离子通道病省部共建教育部重点实验室,广州510260

出　　处：《中华神经医学杂志》2014年第12期1218-1222,共5页Chinese Journal of Neuromedicine

基　　金：广东省自然科学基金（S2011010003392）;广州市属高校科研计划（10A223）

摘　　要：目的探讨激活蛋白-1（he-1）c-Jun与Fral对类多巴胺能神经细胞存活能力的影响。方法（1）体外培养SH-SY5Y细胞，裂解后进行免疫共沉淀实验，检测c-Jun是否与Fral形成二聚体。（2）将细胞分为4组，分别为对照组[加入二甲基亚砜（DMSO）]、SP600125组、U0126组及SP600125＋U0126组[分别加入c-Jun氨基末端激酶（JNK）抑制剂SP600125、MEK1／2抑制剂U0126及SP600125＋U0126]。免疫印迹法检测c-Jun或Fral表达，MTT法检测细胞活力。（3）用滴度为100MOI腺病毒（Ad）感染细胞，共分为4组，分别为对照组（转染绿色荧光蛋白）、Ad-c-Jun组、Ad-Fral组、Ad-c-Jun＋Ad-Fral组（后3组分别转染相应Ad），免疫荧光染色检测感染率，MTT法检测细胞活力。结果（11免疫共沉淀结果显示c-Jun和Fral形成二聚体。（2）免疫印迹结果显示SP600125组c-Jun表达减少，U0126组Fral表达减少。MTT结果显示，4组细胞活力差异有统计学意义（F=16．647。P=0．ooo）。其中对照组与SP600125组、U0126组及SP600125＋U0126组差异有统计学意义B0．05）。（3）过表达c-Jun、Fral后，4组细胞活力差异有统计学意义（F=14．543，P=0．000），其中对照组与Ad-c-Jun组差异无统计学意义（P〉0．05），对照组与Ad-Fral组差异有统计学意义（P〈0．05），Ad-Fral组与Ad-c-Jun＋Ad-Fral组差异有统计学意义（P〈0．05）。结论c-Jun与Fral形成二聚体促进类多巴胺能神经细胞存活。Objective To investigate the roles ofc-Jun/Fral dimer in viability of dopaminergic neurons. Methods （1） Dopaminergic neuron-like cell line SH-SYSY was cultured in vitro. Cells were lysed for immunoprecipitation to determine whether c-Jun dimerized with Fral. （2） Cells received treatments were divided into four groups： Jun N-terminal kinase （JNK） inhibitor SP600125 treatment group, mitogen-activated kinase （MEK） 1/2 inhibitor U0126 treatment group, SP600125＋U0126 treatment group and control group （treated with dimethyl sulfoxide [DMSO]）. Western blotting was performed to detect the c-Jun and Fral expression levels and MTT assay was used to observe the cell viability. （3） Cells infected with adenovirus （Ad） carrying c-Jun or Fral genes were divided into three groups： Ad-c-Jun group, Ad-Fral group and Ad-c-Jun＋Ad-Fral group, and cells transfected with GFP were used as control group; immunofluorescence and MTT assay were performed to determine the infectious rate and the cell viability, respectively. Results （1） Co-immunoprecipitation showed that the c-Jun dimerized with Fral in SH-SYSY cell line. （2） Western blotting indicated that inhibition of JNKby SP600125 reduced c-Jun expression and MEK1/2 by U0126 inhibited Fral expression; MTT assay indicated that there were significant differences in the cellular viability among the four groups （F=I 6.647, P=-0.000）, the cellular viability in control group obviously differed from that in SP600125 treatment group, U0126 treatment group or SP600125＋U0126 treatment group （P〈0.05）. （3） Immunofluorescence showed that most of cells infected by adenovirus expressed c-Jun or Fral. MTT assay indicated that there were significant differences in the cellular viability among the four groups （F=14.543, P=-0.000）, and there were significant differences in their viabilities between control group and Ad-Fral group, and between Ad-Fral group and Ad-Fral＋Ad-c-Jun group （P〈0.05）. Conclusion The c-Jun and Fral forming a di

关 键 词：类多巴胺神经细胞 C-JUN Fra1 存活 

分 类 号：R338[医药卫生—人体生理学]