Sisskin E E, Barrett J C
Cancer Res. 1981 Feb;41(2):593-603.
The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the terminal differentiation of hamster epidermal cells in culture was studied. Epidermal cells were isolated from 1-day-old Syrian hamsters by separating the epidermis from the dermis by cold trypsin treatment. A large number of cells were isolated by this procedure without contamination with dermal fibroblasts. When grown in culture, the epidermal cells divided rapidly, stratified, and differentiated as measured by elaboration of abundant keratin-like amorphous material, red staining with rhodanile blue (which is characteristic of cornifying epithelium), and formation of cornified envelopes. These structures were measured by electron microscopy and quantitation of detergent-insoluble cell ghosts. TPA markedly inhibited this differentiation of the hamster epidermal cells in culture. When grown in the presence of TPA (5 to 1000 ng/ml) for three or more days, the epidermal cell monolayers failed to stain positively with rhodanile blue, and the cell stratification and production of keratin-like material was reduced. The differentiation of the epidermal cells was quantitated by measuring the percentage of cells with cornified envelopes; TPA reduced by up to 70% the number of these terminally differentiated cells. Phorbol didecanoate also inhibited the differentiation of hamster epidermal cells in culture, while phorbol was inactive. The effect of TPA was reversible. When TPA was removed from the media, the cells rapidly differentiated to the same extent as did untreated cells. TPA also stimulated DNA synthesis of the epidermal cells, especially after 10 days in culture when the vast number of cells in control cultures had ceased DNA synthesis. These results are discussed in view of the fact that TPA has not been demonstrated to promote epidermal carcinogenesis in Syrian hamsters.
研究了12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）对培养的仓鼠表皮细胞终末分化的影响。通过冷胰蛋白酶处理将表皮与真皮分离，从1日龄叙利亚仓鼠中分离出表皮细胞。通过该程序分离出大量细胞，且未被真皮成纤维细胞污染。在培养过程中，表皮细胞迅速分裂、分层并分化，这可通过大量角蛋白样无定形物质的形成、罗丹宁蓝红色染色（这是角质化上皮的特征）以及角质化包膜的形成来衡量。这些结构通过电子显微镜和去污剂不溶性细胞空壳的定量分析来测定。TPA显著抑制培养的仓鼠表皮细胞的这种分化。当在TPA（5至1000 ng/ml）存在下培养三天或更长时间时，表皮细胞单层未能被罗丹宁蓝阳性染色，细胞分层和角蛋白样物质的产生减少。通过测量具有角质化包膜的细胞百分比来定量表皮细胞的分化；TPA使这些终末分化细胞的数量减少多达70%。佛波醇十二烷酸酯也抑制培养的仓鼠表皮细胞的分化，而佛波醇则无活性。TPA的作用是可逆的。当从培养基中去除TPA时，细胞迅速分化至与未处理细胞相同程度。TPA还刺激表皮细胞的DNA合成，特别是在培养10天后，此时对照培养物中的大量细胞已停止DNA合成。鉴于尚未证明TPA能促进叙利亚仓鼠的表皮癌发生，对这些结果进行了讨论。
