Haydock P V, Blomquist C, Brumbaugh S, Dale B A, Holbrook K A, Fleckman P
Department of Oral Biology, University of Washington, Seattle 98195.
J Invest Dermatol. 1993 Aug;101(2):118-26. doi: 10.1111/1523-1747.ep12363609.
Ichthyosis vulgaris is an epidermal disorder in which profilaggrin expression is decreased or absent. To determine whether the ichthyosis vulgaris phenotype could be mimicked by eliminating profilaggrin expression, a rat epidermal cell line was transfected with a plasmid that directs the constitutive expression of an RNA that is antisense to normal profilaggrin mRNA. Non-transfected and neomycin-resistant cells not containing antisense plasmid that were grown in the neomycin analogue G418 served as controls. Immunoblot and immunofluorescence analysis showed that profilaggrin protein expression and processing to filaggrin were delayed by 3 to 4 d and decreased in transfected cells. Profilaggrin mRNA was detected in both control and transfected cells only after the cells reached confluence, whereas antisense RNA was detected in transfectants at all times, even prior to confluence. Ultrastructural examination revealed that keratohyalin granules were decreased in number, globular, and heterogeneous in appearance in transfected cells in-contrast to angular structures seen in control cells. Unexpectedly, stratification was impaired, intermediate filaments were noticeably reduced, and cornified cell envelope formation was delayed in transfectants. Unlike ichthyosis vulgaris keratinocytes, where keratin expression is unaffected, appearance of K1 and K10 was delayed and K1/K10 synthesis was delayed and decreased in transfected cells. The precipitous drop in 35S-methionine incorporation into cytoskeletal protein seen at confluence in control cells was delayed by 3 d in transfected cells. We conclude that, rather than producing the ichthyosis vulgaris phenotype, antisense profilaggrin RNA has more broad-reaching effects on in vitro differentiation of rat epidermal keratinocytes.
寻常型鱼鳞病是一种表皮疾病，其中聚角蛋白微丝相关蛋白的表达减少或缺失。为了确定通过消除聚角蛋白微丝相关蛋白的表达是否可以模拟寻常型鱼鳞病的表型，用一种质粒转染大鼠表皮细胞系，该质粒指导合成与正常聚角蛋白微丝相关蛋白mRNA反义的RNA的组成型表达。在新霉素类似物G418中生长的未转染细胞和不含反义质粒的新霉素抗性细胞用作对照。免疫印迹和免疫荧光分析表明，聚角蛋白微丝相关蛋白的蛋白质表达以及向细丝聚集蛋白的加工在转染细胞中延迟了3至4天，并且减少。仅在细胞汇合后才在对照细胞和转染细胞中检测到聚角蛋白微丝相关蛋白mRNA，而在转染子中始终检测到反义RNA，甚至在汇合之前也是如此。超微结构检查显示，与对照细胞中所见的角状结构相反，转染细胞中的透明角质颗粒数量减少、呈球状且外观不均一。出乎意料的是，转染子中的分层受损，中间丝明显减少，角质化细胞包膜形成延迟。与寻常型鱼鳞病角质形成细胞不同，后者的角蛋白表达不受影响，在转染细胞中，K1和K10的出现延迟，K1/K10的合成延迟且减少。对照细胞在汇合时35S-甲硫氨酸掺入细胞骨架蛋白的急剧下降在转染细胞中延迟了3天。我们得出结论，反义聚角蛋白微丝相关蛋白RNA对大鼠表皮角质形成细胞的体外分化具有更广泛的影响，而不是产生寻常型鱼鳞病的表型。
