Cell kinetic study of epidermal keratinocyte

本课题分为以下三个部分进行观察：1)表皮片中角质形成细胞的二维动力学。2)表皮层中干细胞和转运扩增细胞的标记。3) UVB辐射或致癌物对DNA损伤的影响对角质形成细胞动力学的影响。在制作表皮薄片的过程中，我们试图减少基底膜带蛋白的退化。无毛小鼠皮肤最佳浸泡条件为37℃1MNaBr浸泡1小时。用表皮片检测19型角蛋白和b1整合素的表达，这两种蛋白均被认为是干细胞的良好标志物。虽然在部分毛囊中检测到这两种基因的表达，但在毛囊间表皮中几乎检测不到。干细胞也一直被认为是很少分裂的细胞。在伤口修复过程中，干细胞需要合成DNA并进行有丝分裂。由于没有找到适合区分干细胞的标记，我们采用Brdu标记法对DNA合成细胞数量进行计数，其中应该包括干细胞。Brdu阳性细胞多出现在手术创面边缘，与皮肤附属物无特异性定位关系。接下来，我们尝试在DNA合成上调、UVB照射或致癌促进条件下区分终末分化角质细胞和干细胞。两种刺激均引起表皮层基底角化细胞的均匀Brdu标记，分化角化细胞和干细胞之间的标记强度存在差异。表皮片法在表皮分裂和共聚焦显微镜条件较温和的情况下，应成为角化细胞动力学二维评价的有力工具。

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.