CAJ | 학술논문

目的探讨激活蛋白-1 (AP-1)c-Jun与Fra1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Fra1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Fra1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-Fra1组、Ad-c-Jun+Ad-Fra1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 (1)免疫共沉淀结果显示c-Jun和Fra1形成二聚体.(2)免疫印迹结果显示SP600125组c-Jun表达减少,U0126组Fra1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=0.000),其中对照组与SP600125组、U0126组及SP600125+U0126组差异有统计学意义(P＜0.05).(3)过表达c-Jun、Fra1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-Fra1组差异有统计学意义(P＜0.05),Ad-Fra1组与Ad-c-Jun+Ad-Fra1组差异有统计学意义(P＜0.05). 结论 c-Jun与Fra1形成二聚体促进类多巴胺能神经细胞存活.
목적 탐토격활단백-1 (AP-1)c-Jun여Fra1대류다파알능신경세포존활능력적영향. 방법 (1)체외배양SH-SY5Y세포,렬해후진행면역공침정실험,검측c-Jun시부여Fra1형성이취체.(2)장세포분위4조,분별위대조조[가입이갑기아풍(DMSO)]、SP600125조、U0126조급SP600125+U0126조[분별가입c-Jun안기말단격매(JNK)억제제SP600125、MEK1/2억제제U0126급SP600125+U0126].면역인적법검측c-Jun혹Fra1표체,MTT법검측세포활력.(3)용적도위100MOI선병독(Ad)감염세포,공분위4조,분별위대조조(전염록색형광단백)、Ad-c-Jun조、Ad-Fra1조、Ad-c-Jun+Ad-Fra1조(후3조분별전염상응Ad),면역형광염색검측감염솔,MTT법검측세포활력. 결과 (1)면역공침정결과현시c-Jun화Fra1형성이취체.(2)면역인적결과현시SP600125조c-Jun표체감소,U0126조Fra1표체감소.MTT결과현시,4조세포활력차이유통계학의의(F=16.647,P=0.000),기중대조조여SP600125조、U0126조급SP600125+U0126조차이유통계학의의(P＜0.05).(3)과표체c-Jun、Fra1후,4조세포활력차이유통계학의의(F=14.543,P=0.000),기중대조조여Ad-c-Jun조차이무통계학의의(P＞0.05),대조조여Ad-Fra1조차이유통계학의의(P＜0.05),Ad-Fra1조여Ad-c-Jun+Ad-Fra1조차이유통계학의의(P＜0.05). 결론 c-Jun여Fra1형성이취체촉진류다파알능신경세포존활.
Objective To investigate the roles ofc-Jun/Fra1 dimer in viability ofdopaminergic neurons.Methods (1) Dopaminergic neuron-like cell line SH-SY5Y was cultured in vitro.Cells were lysed for immunoprecipitation to determine whether c-Jun dimerized with Fra1.(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 Fra1 genes were divided into three groups:Ad-c-Jun group,Ad-Fra1 group and Ad-c-Jun+Ad-Fra1 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-SY5Y cell line.(2) Western blotting indicated that inhibition of JNK by SP600125 reduced c-Jun expression and MEK1/2 by U0126 inhibited Fra1 expression; MTT assay indicated that there were significant differences in the cellular viability among the four groups (F=16.647,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 Fra1.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-Fra1 group,and between Ad-Fra1 group and Ad-Fra1+Ad-c-Jun group (P＜0.05).Conclusion The c-Jun and Fra1forming a dimer promotes the viability of dopaminergic neuron-like cells.