| Deer antlers are the only known mammalian organs that can periodically regenerate, and can grow rapidly without going cancerous. The biological study about antler regeneration can bring great significance to human organ regeneration and to the prevention and treatment of tumor. It is known that antler regeneration is driven by pedicle periosteum, and pedicle periosteum cells are a kind of antler regeneration stem cells. The Potentiated pedicle periosteum(PPP) can grow and develop into complete antler, but the Dormant pedicle periosteum(DPP) cannot. Thus, the functional difference research between DPP and PPP has become an important way to reveal the mystery of antler regeneration. Membrane is an important barrier to keep various kinds of physiological processes working normally, and has significant functions in immune responses, cells adhesion and migration, cells recognition and signal transduction. Study the membrane proteome of antler stem cells deeply and systematically and it could bring useful information for uncovering the secret of antler regeneration. The in-depth and systematical study in membrane proteome of antlers regeneration stem cells can bring useful information for uncovering the secret of antler regeneration.To investigate the differentially expressed membrane proteins between DPP cells and PPP cells, we used 2D DIGE technology to study the DPP cells and PPP cells and carried out some experiments as follows:(1) Different cell harvest and purification methods were used to extract membrane proteins; Protein assay and SDS-PAGE electrophoresis were used to compare and analyze the different combined methods.(2) We purified membrane proteins with three kinds of methods which included acetone precipitation, 2D Clean up kit and non-treated groups, then analyzed the experiment results.(3) We cultured DPP cells and PPP cells and extracted membrane proteins. After that, we did 2D DIGE experiments and identified interested proteins with mass spectrometry.(4) The different expression membrane proteins were analyzed by bioinformatics, and six proteins were tested by qRT-PCR.Results are as follows:(1) It was better to collect the antler regeneration stem cells in scraping than enzyme-digesting. Compared with acetone precipitation, 2D Clean up kit gained a better 2D electrophoresis pattern, and non-treated group was the worst.(2) The combination of scraping and 2D Clean up kit got an ideal pattern of antler regeneration stem cells membrane proteins.(3) The number of different expression membrane proteins with statistical significance(p<0.05,fold changes≥1.2) was 122 by 2D DIGE, and we identified 53 membrane proteins. 17 of them are up-regulated in PPP cells and 36 of them are down-regulated in PPP cells;(4) The qRT–PCR results of HSPB1, FTL, SEPT11, VCL, RUVBL2 and PHGDH are consistent with 2D DIGE results, which shows the 2D DIGE experiment results are accurate and reliable.Conclusions are as follows:(1) We get the membrane proteins pattern of antler regeneration stem cells, which contains 3398 protein spots.(2) The number of different expression membrane proteins with statistical significance(p<0.05,fold changes≥1.2) was 122, and we identified 53 membrane proteins, which are mainly classified as catalytic activity, catalytic activity, catalytic activity,transporter activity and transporter activity according to molecular function;(3) Among them, 17 membrane proteins are up-regulated in PPP cells, and they are believed in involving in the transformation from DPP cells to PPP cells and candidate proteins of antler regeneration.(4) Vinculin, ANXA5, ANXA2 and HSPB1 are closely related to antler regeneration. Vinculin is a key protein in regulating antler regeneration by effecting the cell migration and cell stability. HSPB1 and some antioxidant proteins may form a redox catalyst balance system to prevent the antler stem cells from translating into tumor cells because of DNA injury, which keeps the rapid growth of the antler without going cancerous. |
