Proteomic Studies Of PD Model Induced By MPP～+ In PC12 Cells

Part I The cytotoxicity of MPP~+ and the construction of the Parkinson's disease modelObjective: To explore the cytotoxicity of MPP~+ and construct the PD model according to the interenational standard. Methods: PC12 cells were treated with different concentrations of MPP~+(50, 100, 200, 250, 300, 500μM) for 12, 24, 48, 72, 96h, respectively. Cell viability was measured by MTT and apoptotic percentage was determined by fluorescent staining. Results: After treatment with 250μM MPP~+ for 48 hour, the PC12 cell viability decreased to 60.14% and the cell apoptotic rate increased to 9.1%. Conclusion: MPP~+ is a cytotoxic substance, which can down-regulate the cell viability and induce apoptosis. Significance: An interentional standard cell model of PD was established, which sets up a reliable basis for the study of MPP~+ mechanisms at proteomic level. Part II Proteomic study of PC12 cell model of Parkinson's disease induced by MPP~+Objective: To explore the mechanism of MPP~+ cytotoxicity at preteome level and find new clues for the pathogensis of PD. Method: PC12 cells were treated with or without 250μM MPP~+ for 48 hours, differential expressions of protein spots from two groups were analysed with two dimensional difference gel electrophoresis(2D-DIGE) , proteins were identified by matrix-assisted laser desorp-tion/ionization-time of flight mass spectrometer(MALDI-TOF MS). Result: About 2000 protein spots were seen in each of 2D-DIGE images. The expressions of 32 proteins were significantly changed in MPP~+ treated PC12 cells compared with the control. Among them, 15 proteins were down-regulated and 17 were up-regulated more than 30%. 7 proteins were identified by MALDI-TOF and can be classied into 5 categories:①proteins with chaperone activity : nascent polypeptide-associated complexα-polypeptide and crystallin, both of them were down-regulated;②cytoskeletal protein: neurofilament light polypeptide, it's expression was up-regulated;③the protein of ERM family, ezrin, which was also up-regulated;④oxidative stress and mitochondrial function related proteins: thioredoxin, mitochondrial processing peptidase, each of them was up-regulated;⑤immunoinflama-tion relaterd protein: complement binder glycoprotein, it's expression was down-regulated. Significance: DIGE and MS technology were used in the investigation the PC12 model of PD induced with MPP~+ for the first time and our findings provide new clues for the etiopathogenesis of PD and the candidates of therapy targets.