Analysis Of Differentially Expressed Proteins Of The Potentiated And Dormant Pedicle Periosteum Cells In Sika Deer(Cervus Nippon) Through 2D-DIGE

Deer antlers are the only known mammalian appendant organs that can periodically regenerate. By histological experiments, we can conclude that the regeneration ability comes from the pedicle periosteum. Antler regeneration is a stem cell-based process. Stem cells locate in the potentiated region of the pedicle periosteum, and they are the basis of deer antler regeneration. The pedicle periosteum is divided into the potentiated region and the dormant region, which are distinguished by the degree of contacting with the velvet skin. The interactions between the potentiated region and its covering skin prompt antler stem cells to develop into a complete deer antler. Therefore, the proteome of pedicle periosteum stem cells may play an important role in the regeneration process. Before, the researches about differential proteomes of the potentiated pedicle periosteum and the dormant pedicle periosteum were mainly qualitative proteomics, such as two-dimensional electrophoresis; there were few reports about quantitative proteomics researches in this field.Firstly, in this study, conditions of two-dimensional electrophoresis(2-DE) of deer antler stem cells were optimized in the following four aspects including cell processing methods, staining methods, isoelectric focusing total volt-hours and protein extraction and purification combination. All these researches could provide a reference for the study of comparative proteomics between the potentiated pedicle periosteum cells and the dormant pedicle periosteum cells through the two-dimensional fluorescence in gel electrophoresis(2D-DIGE), which is a two-dimensional electrophoresis-based technology. Compared with ultrasonic, Bullet Blender had a better effect on cell breaking. It could get more and clearer protein spots by combining colloidal Coomassie brilliant blue staining and silver staining. When the isoelectric focusing total volt-hours were 15000 volt-hours, the gel maps were most suitable for the software analysis. By comparing six kinds of protein extraction and purification combination, the one combining the manual lysate and 2D Clean-up Kit would get more protein spots and clearer map.Then 2D-DIGE was used to separate the protein spots; differentially expressed protein spots were selected by DeCyder 2D(version 7.2). Matrix-associated laser desorption/ionization time-of-flight tandem mass spectrometry(MALDI-TOF-MS) was performed to obtain peptide mass fingerprinting. Mascot software was used to search matched proteins in the NCBI nr databases. We further explored the involved signal pathways of these identified proteins using PANTHER(Protein Analysis through Evolutionary Relationships) and REACTOME analysis. Results showed that there were one hundred fifty-nine differentially expressed protein spots with more than 1.1-fold changes, less than-1.1-fold changes and P values less than 0.05 by the potentiated antler stem cells over the dormant antler stem cells. Among them, 110 were up-regulated and 49 down-regulated. MALDI-TOF-MS identified eighty-four differentially expressed protein spots. Forty-eight of them were positive and belonged to twenty-seven kinds of proteins. The GO analysis and pathway enrichment analysis of the identified proteins were performed.After the comprehensive analysis of the MALDI-TOF-MS data: POTE ankyrin domain family member E, SUMO-activating enzyme subunit 1, Aldolase B, Glucose-regulated peptide 78, Inositol 1,4,5-trisphosphate receptor type 1, Tetratricopeptide repeat protein 36, Histone H4, ATP synthase subunit beta and Vimentin may have intimate relationships with deer antler regeneration, especially the last four proteins which were markers discriminated by DeCyder 2D. These proteins may determine which has the regeneration ability between the potentiated pedicle periosteum cells and the dormant pedicle periosteum cells. In conclusion, antler regeneration is a process from the dormant to the potentiated states in antler stem cells. The process is regulated by multiple proteins and a complicated signal network. This study provides a theoretical foundation for the in-depth exploration of the unique regeneration molecular regulation mechanism of deer antler.