Separation And Culture Of Epidermal Stem Cells And The Study Of Proteomics

Objective To comsummate the fractional cultivation and identification method of epidermal stem cells(ESCs), make preliminary analysis of the differentially expressed proteins between the two types cell and provide clues for studying the proliferation and differentiation of epidermal stem cells in vitro.Method We obtained the single epidermal cell suspension by enzyme digestion and the ESCs were harvested by adherenced rapidly on type IV collagen plate and were cultured in complexed-DMEM medium, the expression of K19,P63,β1 integrin and CD71 were detected in the rapidly adherent cells with immunohistochemical methods and flow cytometry. we detected the cell cycle and observed the reproductive activity by cell growth curve;The protein expression maps of the two types cell were obtained using 2-DE,and the differential expressed protein spots were analyzed by Image Master 2D Elite 5.0 software.Result The rapidly adherent cells grewed well in the complexed-DMEM medium, immunohistochemical showed the rapidly adherent cells were positive for P63 and keratin 19, the express-ratio ofβ1 intergrin and CD71 were 98.57% and 9.05%. Cell cycles showed that about 94.99% cells were in resting state/pre-DNA synthetic gap(G0/G1 phase).The growth-curve showed that the rapidly adherent cells presented exponential growth. These results indicated that the rapidly adherent cells were epidermal stem cells; There were well reproducibility and comparability between the 2-DE protein expression maps of the two cell lines. Average protein spots were 982±18,930±15 in keratinocytes and epidermal stem cells,the matched spots were 850±13,798±11 in both the two types cell, and the average matching rate were 86.56% ,85.81%.There were 886±8 protein spots were matched between the two types cell, the average matching rate was 76.98%. There were 11 differential protein spots were identified between the two types cell,eight protein spots were expressed only or higher in the epidermal stem cells and three protein spots were expressed only or higher in the keratinocytes.Conclusion We confirmed that the rapidly adherent cells were epidermal stem cells,we obtained 2-DE maps of keratinocytes and epidermal stem cells which with high resolution and reproducibility by the technique of two-dimensional gel electrophoresis and there were some differences in protein expression between these two types cell. it was significant for obtaining the seed-cell of tissue engineering skin and exploring the proliferation and differentiation mechanism of epidermal stem cells in vitro.