MPTP作用下小鼠黑质和纹状体中不同时期基因表达变化

Early and Late of Gene Expression Changes in Substantia Nigra and Striatum of MPTP Mice Model Employing Oligo Microarray

目的了解帕金森病小鼠模型黑质和纹状体中差异表达的基因以及在MPTP作用后不同时间点基因表达变化的规律。方法取16只SPF级C57BL/6J小鼠,用数字表法随机分为2组。MPTP组:30mg/kgMPTP腹腔注射1次。对照组:注射等量生理盐水。制模后分别在1d和2个月时分离黑质和纹状体。抽提总RNA,利用寡核苷酸芯片分析1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)作用下不同时间点的差异表达基因。结果所检测的1200个基因中,黑质在制模后第1天有13个基因差异表达,2个月时有6个基因差异表达;纹状体中第1天有12个基因差异表达,2个月时有13个基因差异表达。差异表达的基因与多种生理过程,如应激反应、蛋白质磷酸化、蛋白酶体系统、信号转导等密切相关。结论帕金森病的发生、发展过程涉及到多种基因表达水平和多个生理过程的改变。黑质中γ-氨基丁酸通路以及纹状体中蛋白质的磷酸化水平和正确折叠在MPTP小鼠模型中发生了显著变化。

Objective Parkinson＇ s disease （PD） is a progressive neurodegenerative disorder characteriged by that results in degeneration of nigro-striatal dopamine neurons with the deficiency of dopamine in the striatum. The causes and underlying mechanism for the loss of dopaminergic neurons is still elusive but believed to be associated with alterations of gene expression in both substantia nigra and striatum. The aim of this study is to figure out differently expressed genes in substantia nigra and striatum in experimental parkinsonism, especially expression profiles at different time points post MPTP administration. Methods Sixteen C57BL/6J male mice were divided into 2 groups： MPTP group was administrated with one dose 30 mg/kg MPTP, which induced sub-acute animal model of parkinson＇ s disease. Control group was treated with saline. Mice were separately sacrificed at 1 day post treatment, when acute response was obvious, and 2 months post treatment, when mice underwent a recovery. Total RNA was isolated from substantial nigra or striatum using Trizol reagent. It was then reversely transcribed into cDNA using Oligo dT primer, which served as template for the generation of cRNA. Consequently, cRNA was labeled using Klenow fragment. Then the probes were hybridized to MO2 oligo chips representing 1 200 unique mouse genes. The chips were scanned with a ScanArray 4000. The acquired images were analyzed using GenePix Pro 4.0 software. Locally weighted scatterplot smoothing（LOWESS） normalization was carried out to reduce system biases. Results In substantia nigra, 13 genes expressed differently at day 1, and much less（6 genes） at 2 months. In addition, at acute stage （day 1 ）, most genes with altered expression were stress protein or signal transducer or related to apoptosis, suggesting these pathways were associated with the initiation of loss of dopaminergic neurons in substantia nigra. In supporting this assumption, at recovery stage, most of these genes altered their expression reversely, indicating dopaminergic neurons in substania nigra undergoing a recovery. However, in striatum, 12 genes expressed differently at day 1, and similarly, 13 genes at 2 months. It was likely that differentially expressed genes at acute stage were related to the transient toxicity of MPTP, while those at recovery stage were regulated by the deficiency of dopamine in striatum. Conclusion Our results indicate that oxidative stress has a role in the mechanism of dopaminergic neuron degeneration. It is likely that both endotoxin and extrotoxin will cause the accumulation of reactive oxygen species and lipid oxidation in substantia nigra and then results in the cell loss in substantia nigra. However, the present gene expression analysis has clearly indicated that the process of dopaminergic neurodegeneration is a complex cascade of events that simple oxidative stress can not fully explain. It appears that multiple pathways are involved in the pathogenesis of PD. Gamma-aminobutyric acid pathway is altered in substantia nigra during MPTP treatment. Gammaaminobutyric acid is a repressive neurotransmitter which is abundant in striatum and plays an important role in regulating function of basal ganglia. It appears that dopamine deficiency may modify the synthesis and secretion of gamma-aminobutyric acid in neurons in striatum and subsequently worsen the PD syndrome. Additionally, protein phosphorylation and proper folding may change dramatically in striatum of MPTP mice which might relate with the protein aggregation, a hallmarker of Parkinson＇ s disease. These newly identified genes afford a quantitative view of the changes that accompany PD at the genomic level, and provid a deeper insight into the molecular basis of the disease.