Cell kinetic study of epidermal keratinocyte

点击翻译按钮获取中文摘要

This project is divided in following three parts to observe 1) the kinetic of keratinocyte two-dimensionally in epidermal sheet. 2) the marker of the stem cell and transit amplifying cell in epidermal sheet. 3) the changes in the kinetic of keratinocyte that was affected DNA damage by UVB irradiation or carcinogen. At the start, we tried to minimize the degeneration of basement membrane zone protein during making epidermal sheet. Best condition for hairless mouse skin is soaking in 1MNaBr at 37℃ for 1 hour. The expression of type 19 keratin and b1 integrin, both were reported as good marker for stem cell, was tested with epidermal sheet. Though the expression of both was detected in part of hair follicules, that was hardly detected in interfollicular epidermis. Stem cell also has been thought as rarely dividing cell. During wound repair, Stem cells should synthesize DNA and make mitosis. Since we did not find the suitable marker for distinction of stem cell, we adopted to count DNA synthesizing cell number by Brdu labeling which should include stem cell. Most of Brdu positive cells were found at the edge of surgical wound with no specific localization or relation to skin appendages. Next, we tried to discriminate between terminal differentiating keratinocyte and stem cell under the up-regulating condition of DNA synthesis, UVB irradiation or carcinogenic promotion. Both stimuli caused uniform Brdu labeling throughout basal keratinocyte in epidermal sheet and the difference of labeling intensity between differentiating keratinocyte and stem cell.Epidermal sheet method should be a powerful tool for the two-dimensional evaluation of keratinocyte kinetics with milder condition for epidermal splitting and confocal microscopy.