Proteomics Study In Substantia Nigra Of Chronic Mouse Models Of Parkinson's Disease Induced By Rotenone And MPTP

Objective1.To optimize the two-dimensional gel electrophoresis(2-DE) technique for substantia nigra protein of mouse by comparing the effects of different sample pretreatment methods,different gels and different amount of protein loads to the 2-DE maps.2.To explore the disease-specific proteins of PD by comparing the protein expression profiles of substantia nigra in the chronic mouse models of Parkinson's disease induced by Rotenone and MPTP.Methods1.Extracted the protein of substantia nigra from the normal C57BL mouse, pretreated the protein with the methods of TCA/acetone and clean-up kit then ran the 2-DE with different gels(pH3-10L,pH3-10NL and pH4-7) and different amount (80ug,180ug and 300ug) of protein loads.2.Established the chronic mouse models of Parkinson's disease as rotenone group and it's control group1,MPTP group and it's control group2 respectively,evaluated the behavior changes(open-field test) and immunohistochemistry of Tyrosine hydroxylase(TH).3.Extracted the protein of substantia nigra from rotenone group and control group1,MPTP group and control group2 respectively,pretreated the protein with the methods of clean-up kit and ran the 2-DE with the gel of pH4-7 and the load of 180ug.4.Obtained the differentially expressed proteins through comparing the maps of rotenone group and control group1,MPTP group and control group2 respectively with Pdquest8.0.5.Digested the differentially expressed proteins with trypsin,then identified with MALDI-TOF-MS,searched in database with the obtained Peptide Mass Fingerprint and bioinformation analysis.Results1.The results of 2-DE in substantia nigra of normal mouse:â‘ Compared with clean-up kit,the method of TCA /acetone lost the small and middle molecule weight proteins partially and the yields of which was lower.â‘¡The protein spots of pH3-10L and pH3-10NL were distributed in the middle of the maps.However,the protein spots of pH4-7 were distributed in the map evenly and there are less streaks.â‘¢The backgroud of the map of 80ug was clear,but only 978 protein spots were detected.The map of 180ug was not only clear and with a high-resolution,but 1267 protein spots were also detected.The number of 1371 protein spots were detected in the map of 300ug,but the background of which was dirty and there were many streaks.2.The results of behavior and immunohistochemistry:â‘ Compared with control group1 and control group2, rotenone group and MPTP group displayed significant bradycardia,tremor and decreased and slowly movements,just according to Parkinson's disease-like symptoms.And there are significant different in open-field test(P<0.05).â‘¡Compared with control group1 and control group2,there are significant decrease of positive cells of TH in substantia nigra in rotenone group and MPTP group,and there are significant different(P<0.05).3.The results of 2-DE and MS: Twenty-tuo differentially expressed proteins were found between rotenone group and control group1,while 28 were found between MPTP group and control group2.And 7 differentially expressed proteins were covariant.Three of which were identified successfully:PPP2R1A,Pyridoxal kinase and Peroxiredoxin-2.Conclusion1.After the sample be pretreated with clean-up kit,a high-resolution and clear 2-DE map for substantia nigra protein of mouse can be obtained with pH4-7gel and 180ug protein loads.2. A new research model in proteomics was adoptted in two chronic mouse models of PD induced by rotenone MPTP and three differentially expressed proteins were identified successfully.The changes of those proteins may be related with the pathogenesis of PD and may be the DSPs of PD.