GPX3基因缓解MPP^(+)诱导多巴胺细胞损伤的机制研究

Study on mechanism of glutathione peroxidase 3(GPX3)gene alleviating 1-methyl-4-phenylpyridinium(MPP^(+))induced dopaminergic cell injury

目的 探讨GPX3基因缓解MPP^(+)诱导多巴胺细胞损伤的机制。方法 用Illumina表达谱芯片筛查芯片-PD组(Chip-PD组,6例)帕金森病(PD)患者与芯片-对照组(Chip-对照组,5例)体检健康者的外周血标本中差异基因。然后在人-PD组(Hum-PD组,45例)PD患者和人-对照组(Hum-对照组,47例)体检健康者的外周血中及细胞-PD组(Cel-PD组)和细胞-对照组(Cel-对照组)细胞模型中检测目的基因GPX3的表达差异。最后利用过表达GPX3的病毒转染MN9D细胞,比较MPP^(+)处理后细胞-GPX3过表达组(Cel-GPX3过表达组)和细胞-空载组(Cel-空载组)细胞活力及细胞计数的差异。结果 (1)基因芯片的聚类分析显示30个基因上调,52个下调,其中KEGG富集分析显示GPX3基因表达下调,并可能通过花生四烯酸代谢通路与PD的发病机制有关,差异有统计学意义(FE=11.794,P<0.05)。(2)与Hum-对照组相比,Hum-PD组GPX3基因表达下调,差异有统计学意义(t=-4.738,P<0.001)。(3)Western blot结果显示PD细胞模型中GPX3蛋白相对表达量随着MPP^(+)的处理浓度的增加而逐渐下降,差异有统计学意义(F=21.75,P<0.001)。(4)MPP^(+)处理后,Cel-GPX3过表达组的细胞活力百分比高于Cel-空载组,差异有统计学意义(t=2.659,P=0.011)。且Cel-GPX3过表达组MN9D细胞计数较Cel-空载组增高,差异有统计学意义(t=3.697,P=0.004)。结论 GPX3表达增高可有效缓解MPP^(+)诱导的多巴胺细胞损伤。

Objective Exploring the mechanism of Glutathione peroxidase 3(GPX3)gene attenuating 1-methyl-4-phenylpyridinium(MPP^(+))induced dopamine cell injury.MethodsIllumina expression profile chips were used to screen differential genes in the peripheral blood samples of the patients with Parkinson′s disease(PD)in chip PD group(chip PD group,6 cases)and healthy people in chip control group(chip control group,5 cases).Then the expression of GPX3 was detected in the peripheral blood of PD patients and healthy people in the human PD group(Hum PD group,45 cases)and the human control group(Hum control group,47 cases),and in the cell PD group(Cel PD group)and cell control group(Cel control group)cell models.Finally,mouse midbrain dopaminergic neuron cells(MN9D)cells were transfected with the virus overexpressing GPX3,and the cell viability and cell counts were compared between the Cel-GPX3 overexpression group and the Cel-empty group after MPP^(+)treatment.Results(1)The cluster analysis of the gene chip showed that 30 genes were up-regulated and 52 were down-regulated.Among them,KEGG enrichment analysis showed that the expression of GPX3 gene was down-regulated,which may be related to PD through the arachidonic acid metabolic pathway related to the pathogenesis,the difference was statistically significant(FE=11.794,P<0.05).(2)Compared with the Hum-control group,the expression of GPX3 gene was down-regulated in the Hum-PD group,and the difference was statistically significant(t=-4.738,P<0.001).(3)The results of Western blot showed that the relative expression of GPX3 protein gradually was decreased with the increase of MPP^(+)treatment concentration,and the difference was statistically significant(F=21.75,P<0.001).(4)After MPP^(+)treatment,the percentage of cell viability in the Cel-GPX3 overexpression group was higher than that in the Cel-empty group,and the difference was statistically significant(t=2.659,P=0.011).And MN9D cell count in the Cel-GPX3 overexpression group was higher than that in the Cel-empty group,and the difference was statistically significant(t=3.697,P=0.004).ConclusionIncreased expression of GPX3 can effectively alleviate MPP^(+)induced dopamine cell injury.