| 1 BackgroundEcdysterone (EDS) is one of the most important hormone of two kinds of ecdysone. Ecdysone, which was researched by entomologist in early time, is the hormone which modulates the exuviability of the hexapod. It exists not only in the ecdysis process of the hexapod, but also in others periods. There is much more ecdysterone in many kinds of plants such as ferny, gymnosperm and angiosperm than that in hexapod. It was found in mammalian even humans sometimes.It plays an important role in growth, ecdysis and procreation of invertebrate,especially arthropod.Ecdysterone is indispensable to vertebrate. Many studies indicate that it can promote protein synthesis, modulate glycometabolism, lipid metabolism, gene expression and improve immune function, and it also performs an role in central nervous system, cardiovascular system, proliferation and differentiation of cells, and so on , which is why it was regarded as revive inducer.In the previous studies we found the ecdysterone cream can obviously promote wound healing in rabbits. The evidence is the higher percentage of the healing wound area, the faster healing speed, the better healing quality, the higher capillary vessel density. We protracted the dose-effect analysis curve based on the percentage of the healing wound area. Wound healing is a series of complicated renovation activities by the organism to resume the continuity and integrality, keep the stabilization of the internal environment, which mainly contains a series of complicated biological progresses such as the hyperplasia of the granulation tissue, the repithelization , the reconstruction of the wound healing, and so on. The proliferation of epithelial cell, endothelial cell and fibroblast is the key point of the hyperplasia of the granulation tissue ,so it is also plays an important role in the process of the wound healing. Human epidermal stem cells is the origin of the epidermal cells in wound healing progress. The renovation of epidermal cells mainly result from the proliferation and differentiation of human epidermal stem cells. Considering the role of the repithelization playing in the wound healing, we speculate ecdysterone can promote the proliferation of the human epidermal stem cells, and it is one of the mechanisms why it can promote the wound healing.Moreover, the main effect of the medication used to promote the wound healing at present is to stop bleeding and prevent tissue from swelling and keep the wound, not to promote the cells proliferation. So we designed this study to observe the effect of ecdysterone on the proliferation of human epidermal stem cells, and investigate the further mechanisms to provide further theoretical foundation for the ecdysterone cream made by ourselves to use in future clinical practice.2 ObjectiveTo observe the effect of ecdysterone on the proliferation of human epidermal stem cells, to investigate the further mechanism and provide further theoretical foundation for the ecdysterone cream made by ourselves to use in future clinical practice.3 Methods The method protease-collagenⅣ-coated adhesion was used to isolate human epidermal stem cells from human foreskin,and immunocytochemistry technique was used to identify the third generation by the markers intergrinβ1,keratin 19, keratin 14 and keratin 10.The third generation was used in this study. There were six groups in total. Human epidermal stem cells of 5 experiment groups were respectively cultured in the Epilife with EDS 10mg/L,20mg/L, 40mg/L,80mg/L, 160mg/L,that of control group were cultured in the Epilife without ecdysterone. On the lst,2nd,3rd and 4th day after the human epidermal stem cells were cuntured, the optical density(OD) was measured by method MTT ( 3-(4,5-dimehyl-2-thiazolyl)-2,5-dimehyl-2H-tetrazzolium bromide) to evaluate the effect of ecdysterone on the proliferation of human epidermal stem cells.Meanwhile, the cell cycles of the control and 80mg/L group were determined with flow cytometry to calculate the proliferation index(PI),on the 4th day, PI=(S+G2/M)/(G0/Gl+S+G2/M)×100%. In the statistical analysis, the optical density was represented by Mean±Standard deviation ((?)S).The difference between the 6 groups and the 4 days was analysed in the method analisis of variance of factorial experiment with the software SPSS 13.0. The proportion of G0/G1, S, G2/M stage and the proliferation index was represented by Mean±Standard deviation ((?)S), the difference between the control and 80mg/L group was analysed in the method analisis of independent-Samples T Test with the software SPSS 13.0. The significant standard is 0.05.4 ResultsImmunocytochemistry showed human epidermal stem cells were positive for intergrinβ1,keratin 19 and keratin 14,but negative for keratin 10.Statistical analysis of the optical density showed that the ecdysterone can promote the proliferation of human epidermal stem cells (P<0.05), and its optimum effective concentration was 80mg/L.Flow cytometry showed the lower G0/G1 stage proportion of 80mg/L group than control gtoup, but the higher S , G2/M stage proportion and proliferationindex .ConclusionEcdysterone can promote the proliferation of human epidermal stem cells. The optimal concentration is 80mg·L-1.One mechanism of the promoting effect may be that ecdysterone can promote human epidermal stem cells enter into S stage from G0/G1 stage, and into G2/M stage from S stage.The promoting effect may be one of the mechanisms why it can promote the wound healing.
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