Study On The Location,Culture And The Expression Of The Telomerase Of The Human Epidermal Stem Cell

1 Objectives(1) To study the characteristics of the location and the telomerase expression of the different developmental periods of epidermal stem cells, to illuminate the significance of these characteristics to the burn wound repair.(2) To study the isolation,culture and identification of the epidermal stem cell, to compare the the telomerase expression of the different developmental periods of the epidermal stem cells, to provide seed cells for the skin tissue engineering.2 MethodsPartⅠthe detection of the location and the telomerase expression of the of epidermal stem cellsThe skin samples of child and adult that came from the operation of burn and plastic, the samples of fetus came from the accidental abortion. The samples were fixed by 10% formalin at once, then were made into paraffin sections. Integrinβ1,keratin19,p63 transcription factor and TERT were detected by the immunohistochemistry stain. Optical microscope were used to observe the stain, the buffy expression as the positive, the disposal without the first antibody as the negative control.PartⅡThe isolation,culture and the detection of the telomerase activity expressionThe skin sample of the fetus came from the accidental abortion. The sample was disposed with Trypsin-EDTA and got the epidermis, then digested the epidermis and had the epidermal cells, the typeⅣcollagen was used to isolate and purify the human epidermal stem cells, the epidermal growth factor and the keratinocyte serum free medium were used to culture the cells. Calculate the colony forming efficiency. Theβ1 integrin,k19,p63 and TERT were detected by the immunohistochemistry stain. The cell cycle was determined by the flow cytometer. The epidermal cells were used as the control groups. 3 ResultsPartⅠTheβ1 integrin,K19,p63 and TERT positive cells were mainly located in the epidermal basement membrane, and the expression levels of those indexes were highest in the fetus skin, lower in the skins of the child and adult, the distribution was uniformity in the fetus and adult samples. Lowest expression in the adult sample. The expression amount of theβ1 integrin,K19,p63 and TERT in the different development periods revealed decreasing with the age increasing(P<0.01).PartⅡThe cultured cells revealed colony growth, the colony forming efficiency of the epidermal stem cell is higher than the epidermal cell, P<0.05. the proportion of the epidermal stem cell in G1/ G0 period of the cell cycle was higher than the epidermal cells with the same disposal, P<0.05. Theβ1 integrin,k19 and p63 had positive expression in the epidermal stem cells but not the epidermal cells. the TERT immunohistochemistry stain revealed that the expression level of the fetus epidermal stem cells was higher than that of the adult and the child. The detection of the average value of A and S of each group exhibited that the value decreased with the age. Statistics analysis showed that there were statistic differences among them, P < 0. 05.4 Conclusions(1) The epidermal stem cell of the fetus,child and adult were mainly located in the epidermal basement membrane, the positive expression sector and the expression levels of the telomerase had a same level with the location and positive expression level of the epidermal stem cells. It showed that the quantity and the expression level of the telomerase of the epidermal stem cell may have a relation with the repair ability of the different developmental periods of the skin tissue.(2) The epidermal stem cells separated and purified by the Trypsin-EDTA and typeⅣcollagen revealed colony formation, the cells of G0/G1 in the cell cycle had a higher proportion than the epidermal cell, theβ1 integrin,k19 and p63 had positive expression , the epidermal stem cell had the stem cell traits.(3) The different periods of the skin samples had the telomerase activity, the intensity level was followed as the period of the fetus,the child and the adult. It showed that the induction and the reinforcement of the telomerase activity may have important influence in the maintenance of the self-renewal and proliferation ability of the epidermal stem cell.