| Parkinson's disease (PD) is a kind of common neurodegenerative disorder in the aged whose primary pathologic features are the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the presence of eosinophilic inclusions called Lewy body in the cytoplasm of the spared neurons. The etiology and etiopathogenesis of PD are still confused. With the discovery of genes such asα-synuclein, parkin, DJ-1, UCHL-1 et al ,and with their functions declassified, researchers have now put focus on the ubiquitin-proteasome system (UPS).Growing evidences suggest that the dysfunction of the ubiquitin-proteasome system is involved in the etiopathogenesis of PD. The dysfunction or exhaustion of UPS results in the accumulation of abnormal proteins in cells, the defects of cell function and even their death. Many evidences of UPS dysfunctions are present in familial PD, sporadic PD and several PD models. Thus, UPS dysfunction may be a unifying pathway of various types of PD.Proteomics is a powerful screening method for detecting unexpected alterations in protein expression those may be missed by conventional biochemical techniques. It abandons the traditional research pattern through which a single protein is studied independently. For the first time to study protein composition and regularity in the cells as a whole becomes possible. The new method is becoming important for studying neurodegenerative diseases. From recognition of structural proteins to evaluation of differential expression of functional proteins, this technique develops fast. Though studies on PD have made large progress, the mechanism of PD, the early diagnosis and the effective treatment need to be explored urgently. With the development of proteomics technique, the intervention of proteomics, especially comparative proteomics, brings about new hope in these fields.To explore the mechanism by which UPS is involved in PD etiopathogenesis, differentially-expressed proteins were studied and identifed in the PD model in PC12 cells u sing proteome methods ,in order to further explore etiology and etiopathogenesis of PD, in the protein level providing a new clue for the diagnosis and treatment of PD.In this experiment, natural proteasome inhibitor lactacystin was added to PC12 cells to set up a PD cell model, which simulated the human PD's pathologic features - degeneration of dopaminergic neurons and the presence of eosinophilic inclusions.Treated with different concentrations of lactacystin(0,5,10 and 20μmol·L-1))for 24h , cell viability was measured by MTT assay .Inclusion bodies and the accumulation ofα-synuclein in cytoplasm were observed through H&E staining and immunohistochemistry. AO/EB double staining and electron microscope were also adopted to test apoptosis. Protein of PC12 cells were extracted. Maps of the proteins were established by the immobilized pH gradient two-dimensional electrophoresis. Protein spot detection and matching were performed with an image scanner and analysis software. With the help of matrix-assisted laser desorption/ionization time of flight mass spectrometry, protein spots were identified.The results showed cell viability began to decline significantly at a concentration of 10μmol·L-1 (P<0.01) ,as concentrations grew, cell viability declined in a dose dependent manner (P<0.01). H&E staining showed eosinophilic materials close to nucleus .Immunohistochemistry demonstrated a brown roundα-synuclein immunoactive mass in cytoplasm. As time prolonged and concentration raised, the inclusions began to emerge and transmigrate extracellular gradually. AO/EB staining showed viable apoptotic and electron microscope showed nucleus shrinked and dyssymmetred ,some karyopycnosis and aggregated. There were 46 differentially expressed proteins were found in the PD model with 26 increased and 20 decreased. Mass-spectrum idetifed 6 of them,as Peripherin,Tyrosine hydroxylase(TH),Heat shock protein 5,cysteine proteinase inhibitorb6(Serpinb6),Aldehyde reductase(AR),Heat shock protein 8.In the experiment the PD cell model led by lactacystin in PC12 cells was established . Lactacystin has toxic effect on dopaminergic neurons,which formed inclusion body and lead apoptosis.Ubiquitin-proteasome dysfunction might play an important role in the pathogenisis of Parkinson's disease .Cells with lactacystin can produce cytoplasmic eosinophilic andα-synuclein immunoreactive inclusions . The development of inclusions in this model are consistent with those in human Parkinson's disease and diffuse Lewy body disease and may be helpful to further explore the mechanism of neurodegenerative diseases. Six differently expressed proteins were found in the PD cell model induced by proteasomal inhibition . Peripherin and Serpinb6 were first reported in PD correlated models. All these may be a valuble clue for the exploration of the etiology and etiopathogenesis of PD. |
