Gibbs S, Backendorf C, Ponec M
Department of Dermatology, University Hospital Leiden, The Netherlands.
Arch Dermatol Res. 1996 Nov;288(12):729-38. doi: 10.1007/BF02505289.
We studied the effect of all-trans retinoic acid (all-trans-RA), 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) on proliferation and differentiation of human keratinocytes cultured in a submerged culture system for up to 5 weeks and evaluated changes in cell morphology and in the expression of proliferation- and terminal differentiation-related genes on both the mRNA and the protein levels. Under control culture conditions, the expression of small proline-rich proteins (SPRR1 and SPRR2), involucrin, Ki67 and c-jun reached a maximum after 2 weeks in culture (1 week postconfluence) and then decreased as the tissue architecture of the cultures deteriorated. Upon simultaneous treatment with both retinoids and 1,25(OH)2D3 a culture was generated that remained stable for 4 weeks with at least eight living cell layers. Furthermore, this culture showed a pattern of SPRR2 and involucrin expression which closely resembled that of native epidermis, a maintained Ki67 expression and a strongly induced c-jun expression. Treatment with 1,25(OH)2D3 alone inhibited cell proliferation and stimulated cell differentiation resulting in acceleration of the differentiated phenotype and was accompanied by inhibition of c-jun and Ki67 expression and also, surprisingly by inhibition of SPRR1, SPRR2 and involucrin expression. In contrast, treatment with all-trans-RA and/or 9-cis-RA induced a more proliferative phenotype with a prolonged lifespan as compared to control cultures. SPRR1 was weakly repressed, SPRR2 was strongly repressed, a delayed induction of involucrin occurred, and c-jun and Ki67 expression were maintained. These results show that modulation of the composition of the medium by the addition of various vitamins results in changes in the balance between keratinocyte proliferation and differentiation which correspond to changes in the expression of proliferation and differentiation markers and prolongation of the culture lifespan.
我们研究了全反式维甲酸（all-trans-RA）、9-顺式维甲酸（9-cis-RA）和1,25-二羟基维生素D3（1,25(OH)2D3）对在浸没培养系统中培养长达5周的人角质形成细胞增殖和分化的影响，并评估了细胞形态以及增殖和终末分化相关基因在mRNA和蛋白质水平上的表达变化。在对照培养条件下，富含脯氨酸的小分子蛋白（SPRR1和SPRR2）、内披蛋白、Ki67和c-jun的表达在培养2周后（汇合后1周）达到最大值，然后随着培养物组织结构的恶化而下降。同时用维甲酸和1,25(OH)2D3处理后，产生了一种至少有八个活细胞层且能稳定4周的培养物。此外，这种培养物显示出SPRR2和内披蛋白的表达模式与天然表皮非常相似，Ki67表达维持不变，c-jun表达强烈诱导。单独用1,25(OH) D3处理可抑制细胞增殖并刺激细胞分化，导致分化表型加速，同时伴有c-jun和Ki67表达的抑制，令人惊讶的是，还伴有SPRR1、SPRR2和内披蛋白表达的抑制。相比之下，与对照培养物相比，用全反式维甲酸和/或9-顺式维甲酸处理诱导了一种增殖性更强、寿命延长的表型。SPRR1受到弱抑制，SPRR2受到强抑制，内披蛋白的诱导延迟，c-jun和Ki67表达维持不变。这些结果表明，通过添加各种维生素来调节培养基的成分会导致角质形成细胞增殖和分化之间的平衡发生变化，这与增殖和分化标志物的表达变化以及培养寿命的延长相对应。
