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

反义RNA抑制类固醇硫酸酯酶基因表达：X连锁鱼鳞病细胞模型的建立

基本信息

批准号：
05670745
负责人：
MANABE Motomu
金额：
$ 1.41万
依托单位：
Juntendo University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

由于我们的最终目标是建立先天性皮肤病的细胞模型，因此利用适当的培养系统至关重要。因此，在本项目中，我们尝试建立在癌症研究和发育生物学中被广泛研究的多细胞球体方法。正常人表皮角质形成细胞与成纤维细胞在培养皿上共培养，培养皿上涂有热响应聚合物，聚n -异丙基丙烯酰胺（PNIPAAm），详见其他地方。随后，角化细胞附着的成纤维细胞单层从PNIPAAm胶原基质上完全分离，形成自支撑层。第四天结束时，可以看到5-7层。在球形培养体系中。未见明显的角化标志，如角质透明素颗粒和致密的角质层。随着时间的推移，可见一些核固缩改变和核缺失。虽然我们的模型在体内没有显示出与表皮相同的形态，但球体细胞的特征支持球体是反映活体器官中细胞在体内状态的良好体外实验模型。此外，我们尝试通过电穿孔法将基因转染到角质形成细胞中。作为初步研究，我们将头发角蛋白基因转染到COS-1细胞中。利用毛发角蛋白单克隆抗体作为标记，我们成功检测了COS-1细胞中毛发角蛋白的表达。然而，这种条件不适合角化细胞，因为角化细胞的活力在电穿孔后显著下降。我们一直在寻找合适的条件来培养角质形成细胞，因此在不久的将来，转染效率将会提高。