| Sleep is one of the basic human needs and sleep quality is critical for daily functions of human. For the rapid development of world, sleep deprivation has been an epidemic phenomenon in modern Society. It has generally been observed to have detrimental effects on nervous system, digestive system, cardiovascular system and the immune system, especially in nervous system, symptoms arising from sleep deprivation include impaired cognitive ability, reduced vigilance, mood changes. However, it remains unknow how sleep deprivation affects the body, which limit the established of molecular targets and the discovery of specific drugs and methods.Sleep deprivation can be induced by platform technique, drug admition, forced locomotion technique, gental handling, et al. Among them platform technique is a classical method. Previous studies have showed platform technique can be observed animal weight loss, memory deficits, decreased immunity, REM sleep time is increased and wake time is decreased, and after sleep rebound REM sleep time and wake time is normal. So platform technique is a better method to esterblish REM sleep deprivation model. In our study, we esterblished wistar rats’ REM SD model by modified multiple platform technique.The change of serum protein spectrum is one of the most direct consequences of many diseases. Serum proteomics plays an important role in the research of pathophysiology, the discovery of molecular drug targets and specific markers for early diagnosis. So our research, using the REM sleep deprivation model, sdudy the sleep deprivation rats’ serum proteomics.Hower, proteomic analysis of serum has often been hampered by the high abundance proteins which result in the decrease of the resolution. Therefore, how to remove high abundance proteins and increase the proportion of low abundance proteins is the most important problem faced. So we first systematically investigated the removal of serum high abundance proteins by organic solvent and esterblish the optimization methods. Finally, we study the differentely expressed proteins of REM SD rats’ serum by using serum comparative proteomics methods.(1) Generaol condition: Compared with the Blank Control group and Model Control group, Model group were found irritability and pelage is dull, while no changes in the weight and ability of learning and memory were observed.(2) Established and optimized the method of serum proteomics: Different experiments were systematically designed and performed to investigate the best conditions for 2-DE. Bradford method was used to measure the total serum proteins content, SDS-PAGE was used to evaluate the removing efficiency for high abundance proteins in serum, and the optimized experiment conditions were validated by 2-DE. Date showed that organic solvent, consist of TCA and acetone(1:9, v/v), 4 times of serum volume, had the best efficiency for removing high abundance proteins. For the protein precipitation washed by acetone, 3 times and 1.5 ml per times is the optimal condition. The isoelectric focusing electrophoresis was also optimized. the optimal condition is that Samples containing about 1mg of proteins were dissolved in rehydration buffer, and isoelectrically focused at 50 V for 16 h, 250 V( linear) for 30 min, 1000 V( rapid) for 1 h, 10000 V( linear) for 5 h, 10000 V( rapid) for a total of 70000 Vh, and 1000 V( rapid) for 36 h. Compared with untreated serum, the 2-DE maps shows optimal method can remove high abundance proteins more efficiency and specificity.(3) Differentially expressed proteins: Protein spots mainly distributed in the molecular mass range between 20-95 KDa and the isoelectric point range between 4.5 and 6.5. There was no significant difference among three groups in the total number of spots, the total number of matched spots, the percentage of matched spots relative to the total number of spots on the gel and the percentage of matched spots on the gel relative to the total number of spots on the master. Compared with the Blank Control group and Model Control group, there are at lest two proteins were down regulated in the region where the molecular mass range between 10-30 KDa and the isoelectric point range between 5.5-6.0. In the region where the molecular mass range between 30-50 KDa and the isoelectric point range between 5.5-6.5, 4 proteins were down regulated compared with the Blank Control group and Model Control group; In the region where the molecular mass range between 50-80 KDa and the isoelectric point range between 5.5-6.0, 2 proteins were up regulated compared with the Blank Control group and Model Control group.(4) Mass spectrometric identification and Bioinformatics analysis of differentially expressed proteins: Six spots were identificated by LC-MS/MS. the spots SSP4310, SSP4314, SSP5003, SSP5101, SSP6206 and SSP7201 was serotransferrin, serotransferrin, Glutathione peroxidase 3, Ig kappa chain C region,B allele, collagen alpha-2(I) chain and collagen alpha-2(I) chain, respectively. The down-regulation of serotransferrin which was one of negative acute phase proteins may reduces antioxidant capacity and cause oxidative damage; The down-regulation of glutathione peroxidase 3 implicated that the antioxidant capacity is reduced; The down-regulation of Ig kappa chain C region, B allele implicated the decline in immunity; The down-regulation of collagen alpha-2(I) chain may be related to cardiac injury.In conclusion, we esteblished and optimized the method of proteomics of Wistar serum. Among the 8 proetins which were found changed expressed after 24 h sleep deprivation by using serum comparative proteomics methods search, 2 proetins were up-regulated and 6 proetins were down-regulated. The mass spectrometry identification and bioinformatics analysis were operated, and confirmed two dominant pathogenic mechanisms of Oxidative damage and immune may related to SD. These results could serve as the basis for further proteomic investigations in SD. |
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