Suppression of gene expression of steroid sulfatase by antisense RNA : Establishment of cellular model for X-linked ichthyosis

由于我们的最终目标是建立一个先天性皮肤病的细胞模型，因此使用合适的培养系统至关重要。因此，在本项目中，我们尝试建立多细胞球体方法，该方法已被广泛研究用于癌症研究和发育生物学。将正常人表皮角质形成细胞与成纤维细胞在涂有热响应聚合物聚-N-异丙基丙烯酰胺（PNIPAAm）的培养皿上共培养，如在别处详细描述的。随后，角质形成细胞附着的成纤维细胞单层从PNIPAAm胶原基质中彻底分离，作为自支撑片。第4天，可见5-7层。在球状体培养系统中，未观察到角质化的显著标志物，如透明角质颗粒和致密角质层。随着时间的推移，核固缩和核缺失。虽然我们的模型没有显示出与体内表皮相同的形态，但球体中细胞的特征支持球体是反映活器官中细胞体内状态的良好体外实验模型的观点。此外，我们还尝试通过电穿孔法将基因导入角质形成细胞。作为初步研究，我们将人头发角蛋白基因转染COS-1细胞。以抗毛发角蛋白的单克隆抗体为标记，成功地检测到COS-1细胞中毛发角蛋白的表达。然而，这种条件不适合角质形成细胞，因为角质形成细胞的活力显着下降后电穿孔。我们将继续探索适合角质形成细胞的转染条件，以期在不久的将来提高转染效率。

Since our final goal if to establush a cellular model for a congenital skin disease, it is crucial to utilize a proper culture system. Therefore, in this project, we attempted to establish the multicellular spheroid method which has been widely studied for cancer research and developmental biology. Normal human epidermal keratinocytes were co-cultured with fibroblasts on the dishes coated with a thermo-responsive polymer, poly-N-isopropyl acrylamide (PNIPAAm) as described in detail elsewhere. Later, the keratinocyte-attached fibroblast monolayrs were thoroughly detached from the PNIPAAm collagen substratum as a self-supporting sheet. By the end of fourth day, 5-7 layrs were seen. In spheroid culture systems.salient markers of keratinization such as keratohyalin granules and a compact stratum corneum were not seen. As dayspassed, some pyknotic changes and deletion of nuclei at the core was seen. Although our model does not show the same morphology as shown in epidermis in vivo, the characteristics of cells in the spheroid support the notion that spheroid is a good in vitro experimental model reflecting the in vivo status of cells in living organs. Moreover, we attempted to transfect genes into keratinocytes by the electroporation method. As a preliminary study, we transfected a hair Keratin gene into COS-1 cells. Using a monoclonal antibody to hair keratins as a marker, we could successfully detected the expression of hair keratins in the COS-1 cells. However, this conditions were not suitable for keratinocytes, since the viability of keratinocytes decreased remarkably after electroporation. We keep investigating to find a suitable condition for keratinocytes, therefore the transfection efficiency will be improved in near future.