Research for establishment of evaluation method for the skin irritation using a Three-Dimensional Cultured Human Skin Model instead of in vivo results.

本文以氯化十六烷基吡啶（cetylpyridinium chloride， CPC）为模型试验化合物，分析了MTT法测定人皮肤三维培养模型和无毛小鼠皮肤皮肤刺激的时间过程以及皮肤刺激与刺激浓度的关系。1)皮肤刺激的反应部位虽然体内施用相同剂量的CPC后，背部和腹部皮肤的死细胞数量不同，但对皮肤的CPC浓度有相似的值，说明皮肤刺激与皮肤刺激部位的CPC浓度有直接关系。在用Sigmoid Emax模型评估体外数据时，外形因子Y和最大值Emax与体内研究非常相似。因此，培养的细胞可以用作预测皮肤刺激的工具。2) CPC对角质形成细胞和成纤维细胞的刺激评价皮肤刺激研究显示，死亡的人角质形成细胞的数量是死亡的人成纤维细胞的10倍。这一结果表明，与存在于皮肤深层的成纤维细胞相比，构成皮肤最外层的角质形成细胞在保护机体免受外界刺激方面发挥了重要作用。

In the present report, time course of skin irritation determined by MTT assay and the relationship between skin irritation and the irritant concentration in the three-dimensional cultured human skin model as well as hairless mouse skin were analyzed using cetylpyridinium chloride (CPC) as a model test compound.1)Reaction site of the skin irritationAlthough different numbers of dead cell were found between the dorsal and abdominal skins after in vivo application of the same dose of CPC, a similar value was obtained against the skin concentration of CPC, suggesting that skin irritation is directly related to the CPC concentration where skin irritation takes place. When evaluating the in vitro data by Sigmoid Emax model, form factor, Y and the maximal value, Emax, were very similar to those in the in vivo study. Thus, the cultured cells can be used as a tool for prediction of skin irritation.2)Evaluation of CPC irritation for keratinocytes and fibroblastsSkin irritation study showed number of dead human keratiocytes was 10 times higher than that of dead human fibroblasts. This result suggests that keratinocytes, constituting the outmost layer of the skin, play an important role to protect body from the outside irritation compared to fibroblasts which were existed in deep site of the skin.