关键词: 寡核苷酸微阵列技术, 黑质, 纹状体, MPTP

Abstract: 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. Gamma-aminobutyric 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.

Key words: oligo microarry, substantia nigra, striatum, MPTP