Varani J, Nickoloff B J, Dixit V M, Mitra R S, Voorhees J J
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109-0602.
J Invest Dermatol. 1989 Oct;93(4):449-54. doi: 10.1111/1523-1747.ep12284020.
Human epidermal keratinocytes were established in culture using a low-Ca2+ (0.15 mM), serum-free keratinocyte growth medium (KGM) as the culture medium. Early passage keratinocytes (i.e., between passages 3-8) were incubated for 1 or 2 d in KGM, in KGM supplemented with 1.4 mM Ca2+, or in growth factor-deprived keratinocyte basal medium (KBM). The cells were concomitantly treated with all-trans retinoic acid (0.1-2.5 micrograms/ml), and cell growth was quantitated at the end of the incubation period. The keratinocytes were simultaneously examined for adhesiveness and production of two extracellular matrix molecules, e.g., thrombospondin (TSP) and fibronectin (FN). Treatment with all-trans retinoic acid inhibited proliferation of keratinocytes that were rapidly growing in KGM. Proliferation was also inhibited in KGM supplemented with 1.4 mM Ca2+, but all-trans retinoic acid did not reverse the morphologic features associated with differentiation induced by high Ca2+. In contrast to these effects, all-trans retinoic acid treatment of keratinocytes in KBM, in which the cells were normally quiescent, stimulated growth. In the presence of optimal concentrations of all-trans retinoic acid (0.5 microgram/ml), the rate of keratinocyte proliferation in KBM was approximately 35% of the rate obtained in KGM (maximal proliferation rate). Keratinocyte adhesion (resistance to trypsin-mediated release from the substrate and attachment to the substrate) was inhibited by all-trans retinoic acid under all three conditions. In regard to extracellular matrix production, TSP production was inhibited by greater than 90% under all three conditions in the presence of all-trans retinoic acid. FN production was also inhibited but to a lesser degree. Concentrations of all-trans retinoic acid required to maximally inhibit keratinocyte adhesion and matrix production were higher (1.0-2.5 microgram/ml) than the concentration required to stimulate proliferation in KBM. These in vitro observations may have implications in the effects of retinoids on intact skin, including enhanced keratinocyte proliferation and thickening of the epidermis after topical application to photoaged skin and inhibition of proliferation and cell-cell cohesion after systemic administration in cases of psoriasis.
使用低钙（0.15 mM）无血清角质形成细胞生长培养基（KGM）作为培养基，在培养中建立人表皮角质形成细胞。早期传代的角质形成细胞（即第3 - 8代之间）在KGM、补充有1.4 mM钙的KGM或生长因子缺失的角质形成细胞基础培养基（KBM）中孵育1或2天。细胞同时用全反式维甲酸（0.1 - 2.5微克/毫升）处理，并在孵育期结束时定量细胞生长。同时检查角质形成细胞的黏附性以及两种细胞外基质分子即血小板反应蛋白（TSP）和纤连蛋白（FN）的产生。用全反式维甲酸处理可抑制在KGM中快速生长的角质形成细胞的增殖。在补充有1.4 mM钙的KGM中增殖也受到抑制，但全反式维甲酸并未逆转与高钙诱导的分化相关的形态学特征。与这些作用相反，在正常情况下静止的KBM中用全反式维甲酸处理角质形成细胞可刺激生长。在全反式维甲酸的最佳浓度（0.5微克/毫升）存在下，KBM中角质形成细胞的增殖速率约为KGM中获得的速率（最大增殖速率）的35%。在所有三种条件下，全反式维甲酸均抑制角质形成细胞的黏附（对胰蛋白酶介导的从底物释放的抗性以及对底物的附着）。关于细胞外基质的产生，在全反式维甲酸存在下的所有三种条件下，TSP的产生均被抑制超过90%。FN的产生也受到抑制，但程度较小。最大程度抑制角质形成细胞黏附和基质产生所需的全反式维甲酸浓度（1.0 - 2.5微克/毫升）高于在KBM中刺激增殖所需的浓度。这些体外观察结果可能对视黄醇对完整皮肤的作用有影响，包括局部应用于光老化皮肤后角质形成细胞增殖增强和表皮增厚，以及在银屑病病例中全身给药后增殖和细胞间黏附的抑制。
