EPIDERMAL STEM CELL AND ITS CLINICAL APPLICATION

为了鉴定干细胞，高β 1整合素被用作标记物，但干细胞最重要的特征是其增殖能力。因此，我们测试了角质形成细胞能够增殖多久。从15名年龄在1至70岁之间的健康志愿者获得的角质形成细胞继续培养，直到它们达到衰老。群体倍增时间（PD）范围广泛，为5.7至45.2。这表明干细胞的数量因供体而异。年轻供体的PD倾向于更高，但来自45岁女性的一个菌株的PD为41.3。根据菌株的PD将其分为短、中、长3组。我们认为，这将是可能的，更精确地分析表皮干细胞使用这种分类。三维重建皮肤的制备：将来自人皮肤的成纤维细胞培养在胶原凝胶中，然后将角质形成细胞接种在凝胶的顶部。当角质形成细胞达到汇合时，将共培养物在气液界面下孵育。分层的三维重建皮肤进行了分析，通过免疫化学或电子显微镜。细胞粘附分子和分化标志物在三维重建皮肤中得到充分表达。基底膜成分如半桥粒、大疱性类天疱疮抗原和β 4整合素等形成并充分表达，但锚定纤维或致密层发育不良。此外，我们使用复制缺陷型腺病毒载体将外源基因导入角质形成细胞。β-半乳糖苷酶几乎在所有角质形成细胞中检测到。在三维重建皮肤中，5天后β半乳糖苷酶在表皮中表达，但其在上层中的表达高于基底层。在MOI 10以下未发现毒性。

To identify the stem cell, high beta 1 integurin was used as a marker, but the most important characteristics of stem cell is capacity of its proliferation. So, we tested how long keratinocytes are able to proliferate. Keratinocytes obtained from 15 healthy volunteers ranged between 1 to 70 years old continued to be cultured until they were reached to senescence. Population doubling time (PD) was ranged widely from 5.7 to 45.2. This indicated the number of stem cell was different by donor. PD tended to be higher in younger donor, but PD of one strain from 45 years old female was 41.3. These strains were divided into three groups such as short, intermediate and long, based on their PD. We suggest that it will be possible to analyze epidermal stem cells more precisely using this classification. Preparation of three dimensional reconstituted skin : Fibroblasts from human skin were cultured in collagen gel, then keratinocytes were seeded on the top of the gel. When the keratinocytes reached to confluent, the co-culture was incubated under air liquid interface. Stratified three dimensional reconstituted skin were analyzed by immunohistochemically or by electron microscopy. The cell adhesion molecules and differentiation markers were fully expressed in the three dimensional reconstituted skin. Basement membrane components as hemidesmosome, bullous pemphigoid antigen and beta 4 integurin were formed and expressed sufficiently, but anchoring fibrils or lamina densa were poorly developed. Moreover, we transfer foreign genes into keratinocytes using replication-deficient adenovirus vector. Beta galactosidase was detected in almost all keratinocytes. In the three dimensional reconstituted skin, beta galactosidase was expressed in the epidermis after 5 days, but its expression was higher in the upper layer than the basal layer. The toxicity was not found up to MOI10.