Study On Culture System Of Rat Epidermal Stem Cells And Construction Of Tissue-engineering Skin

Chapter 1-Isolation,culture and identification of rat epidermal stem cells in vitroObjective:To establish a simple and reliable method for separating rat epidermal stem cells from skin in vitro and provide more information for study proliferation and differentiation of epidermal stem cell.Methods:Twenty 1-3 days-old rats of clean grade were selected to prepare epidermal stem cells.Single epidermal stem cell suspension were obtained by enzyme digestion. The target cells were harvested by rapidly adherence on typeâ…£collagen plate and were cultured in complex DMEM containing fetal bovine serum(FBS),0.05 mmol/L CaCl2,epidermal cell growth factor,adenosine,insulin,hydrocortisonum and choleratoxin,ect.Volume percentage of FBS was 20%to maintain call activity when cells were primarily isolated and was 10%when the medium was changed at the second time.Cell growth was observed under light microscope.Keratin 19,Keratin 15 and P63 integrin of separated cells were detected by immunohistochemistry.β1 integrin and CD71,CD34were measured by flow cytometry.Proliferation activity was examined by cell cycle and growth curve.Results:The typeâ…£collagen screened cells grew well in the complex DMEM, resulting in a cell activity of over 98%.Six days later,a clone containing 100-200 cells was detected,showing cobble-stone-like.The rapidly adherent cells were positive for P63,keratin 19and keratin 15.The expression rates ofβ1 intergrin and CD71 were respectively 98.59%and 9.05%.Cell cycles showed that about 94.8% cells were in resting state/pre-DNA-synthetic gap(G0/G1 phase).The growth-curve showed that the rapidly adherent cells presented exponential growth.Conclusion:Rat epidermal stem cells were successfully collected by enzymatic digestion and rapidly adherence on typeâ…£collagen.Complex DMEM containing choleratoxin,adenosine and insulin can maintain proliferation activity of epidermal stem cells.Suitable serum and Ca²⁺ concentrations are also important.Chapter 2-Effects on proliferation and differentiation of rat epidermal stem cells cultured in different system conditionsObjective:To explore the effects on proliferation and differentiation of epidermal stem cells cultured in different system and establish a culture system which can regulate and control the proliferation and differentiation of epidermal stem cells.Methods:The rat epidermis stem cells isolated based on rapidly adherent on typeâ…£collagen cultured in three different system conditions as follows,cultured in common glass dish,cultured with chitin membrane,seeded on the chitin membrane functioning as a cell carrier and implanted subcutaneously into nude mice.Cell colonies were examined under the inverted microscope and the growth of the stem cells on the chitin was observed by the fluorescence microscope and the scanning electron microscope.The colony forming efficiency(CFE) after cultured 4 weeks in these two culture system were measured,the proliferation ability of the epidermal stem cells implanted into mice were evaluated by immunohistochemistry.Furthermore,the influence of the bionic chitin membrane leaching solution to cells was also detected.Results:The separated epidermal stem cells grew well cultured in vitro.The cell began to clonal expansion 3^rd day and confluent dish in 12 days.The proliferation ability gradually decreased during serial subculture and lost after 5-time passage culture in common glass culture dish.Cultured in chitin membrane.,checkerboard cell colonies were visualized on the chitin membrane in 2-4 weeks and massive stem cell colony multiplication was observed under the fluorescence microscope and the scanning electron microscope.The colony forming efficiency after cultured 4 weeks in chitin membrane was also higher than that in culture dish.The stem cell proliferated large amount and formed "epidermal nest" after implanted subcutaneously into nude mice.The chitin membrane leaching solution showed slight cell proliferation at 1:8-1:512 of leaching diluted solution.F=0.781,P＞0.05.The statistics had showed non-significance difference.Conclusion:Epidermal stem cells could have good proliferation ability for a long time cultured with chitin membrane and could be used as seed cells for tissue engineering-skin.It provides evidence for further more animal experiments.Chapter 3-Comparative proteomic analysis of rat epiidermal stem cells and keratinocytesObjective:To study the differential proteomics expression of rat epidermal stem cells and keratinocytes and provide clues for regulating the proliferation and differentiation of epidermal stem cells in vitro.Methods:The single epidermal cells suspension were obtained by enzyme digestion and the epidermal stem cells or keratinocytes were harvested based on rapidly adherence on typeâ…£collagen.Cell total protein were extracted and the concentration were measured according Bradford method.The protein expression maps were presented by two-dimensional electrophoresis(2-DE) and the differential expressed protein spots were analyzed by Image Master 2D Elite 5.0 software.The different-expressed protein spots were detected by matrix assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS) after in-gel protein digestion,Proteins were identified by searching the peptide mass profiles in a public available NCBInr databases(http://www.matrixscience.com).Results:There were good reproducibility and comparability between two cell lines from the 2-DE protein expression maps,Average protein spots were 982±18 in keratinocYtes and 930±15 in epidermal stem cells,the matched spots were 850±13 and 798±11 in these two types cell,and the average matching rate were 86.56%and 85.81%.There were 886±8 matching protein spots between the two types cell,and the average matching rate was 76.98%.Eleven differential protein spots were identified between two type cell.Eight protein spots were only or higher expressed in epidermal stem cells and three protein spots were only or higher expressed in keratinocytes. There were 11 significant proteins successfully identified by MALDI-TOF-MS. Among the 11 proteins,8 proteins were only or higher expressed in epidermal stem cell,including Rho GDP dissociation inhibitor(GDI)) alpha,Translation elongation factor-1,PCNA,GSTM-2,Aldose reductase,ect,whereas 3 proteins only or higher expressed in keratinocyte,including Nitrilase homolog 2,Annexin A5 and Ubiquitin,protein hydrolase,ect.Conclusion:There were some differences in protein expression between epidermal stem cell and keratinocyte,these different protein may be related to the ability of proliferation and differentiation of two type cell.Chapter 4-Repair of full-thickness skin defect wounds in nude mice with engineering-skin composite of epidermal stem cells as seed cells Objective:To study the feasibility of constructing the tissue-engineering skin composite of epidermal stem cells as seed cell and repairing the full-thickness skin defect wounds in nude mice.Methods:Type I collagen were extracted from rat tail tendon with acetic acid and then mixed with chondroitin-6-sulfate to form a collagen-chondrointin-6-sulfate membrane and also demonstrated the membrane "artificial dermis".The tissue-engineering skin was constructed by seeding epidermal stem cells isolated by rapidly adherence on typeâ…£collagen on the artificial dermis substrate and then grafted On the full-thickness skin defect wounds in nude mice.The grafted wounds were observed daily and the specimens were harvested on the 1 to 10 weeks after grafting for histological examination.Results:The artificial dermis was a semitransparent membrane and with a network structure,the pore size was about 50 to 100um.The tissue-engineering skin achieved good adherence to full-thickness defect wounds on the 3 rd day of grafting and better on the 7rd day.The wounds healed about 14 days after grafting and had a good contour of skin with less contraction and scar formation after 10 weeks of grafting. Histological examination showed the epidermal stem cells proliferated promptly and formed 4-5 cell layers after grafting of 14 days and 8-10 cell layers of 6 weeks,large amount of fibroblasts,capillary vessels and little inflammation were observed in the dermis.Conclusions:Tissue-engineering skin composite of epidermal stem cells as seed cell and collagen-6-sulfate had potential prospects in repairing full-thickness skin defect wounds with advantage of good proliferation.