| Background:Coverage of the major severe burn wounds is one of the most difficult problems in front of burn medicine. Autoskin. or cultured epidermis are usually short of supply in patients with major burns on one hand ,and on the other hand ,rejection reaction has not been overcomed if alloskin or heteroskin is used. Therefore, it still remains a major problem to be solved for the coverage of wounds in patients with major burns. Because epidermis derives from epidermal stem cells in the basal cells, it would be possible to bring about a technology revolution in the field of burn wound coverage if we can isolate epidermal stem cells and make them to become the ideal seed cells for construction of tissue engineering skin in vitro by inducing and regulating the differentiation of these stem cells. Objectives:1.To establish the methods for harvesting , identifying and culturing human basal cells.2.To isolate and identify human epidermal stem cells from the basal cells by flow cytometry and the characteristics of these stem cells.3.To lay a foundation for regulating differentiation and growth of epidermal stem cells and for constitution of tissue engineering skin Methods:1.Isolation and identification of basal cells: first of all,. the basal cells in the foreskin were isolated by Dispase I and trypsin; then the cells wereidentified by S-P Method of immunocytochemistry, the mouse anti human k14IgG monoantibody specific to the basal cells was used as the first antibody.2.Identification of epidermal stem cell from basal cells. S-P Method of immunocytochemistry was adopted and the mouse anti human K19IgG monoantibody specific to the epidermal stem cells in keratinocytes were used as the first antibody .3.Culture of basal cells: In the first week , basal cells were cultured on 3T3 feeder layer in DMEM containing 20ng/ml of epidermal growth factor, 0.4 g/ml of hydrocortisone, 10ng/ml of cholera toxin and 10-15% fetal calf serum at 37 and 5%CO2 ; after feeder layer was removed by EDTA , these cells were culture for another 3 ~5 days.4.Isolation of epidermal stem cells: Basal cells were marked by mouse anti human monoantibodies to a 6 3 4 intergrin(CD49f) and goat anti mouse second antibody conjugated at FITC. Then epidermal stem cell and transit amplifying cell subpopulation could be received by flow cytometry; in the end, by its characteristics of adhesion to human placented type IV collagen, epidermal stem cell could be isolated from the subpopulation.5.Identification of epidermal stem cells: The identification was finished by using S-P Method of immunocytochemistry., the first antibodies were mouse anti human K19IgG. Results:1.Basal cells isolated from foreskin by dispase-1 and trypsin had a good viability. Most of the basal cells were round in shape and ahnost all of them were stained with grown-yellow. Nuclei of these cells were large and located in the center.2.There were only a few epidermal stem cells in the isolated basal cells and these cells were stained with grown-yellow. In the meantime, the other basal cells were unstained.3.Basal cells were cultured on human fibroblast feeder layer in DMEM containing fetal bovine serum and epidermal growth factor . For a week or so, most of feed layer cells could be removed with 0.02%EDTA; another 3-5 days later, keratinocytes could be passaged.4.Keratinocyte stem cell and transit amplifying cell population were firstly received from basal cells by flow cytometry after the basal cells were marked with antibodies conjugated at FITC; then the population of cells were cultured in plastic well coated with 100 g/ml human placented type IV collagen. Epidermal stem cells adhered quickly to the plastic dish in 20 minutes, however transit amplifying cells could not adhere to the dish in 60 minutes. Therefore, epidermal stem cells could be received after washing the dish with PBS in 20 minutes.5.Immunocytochemistry showed that the difference in the size of these cells is not obvious. Nearly all of the epidermal stem c...
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