Vicanová J, Boelsma E, Mommaas A M, Kempenaar J A, Forslind B, Pallon J, Egelrud T, Koerten H K, Ponec M
Department of Dermatology, Leiden, The Netherlands.
J Invest Dermatol. 1998 Jul;111(1):97-106. doi: 10.1046/j.1523-1747.1998.00251.x.
Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.
钙在调节表皮角质形成细胞的细胞分化和脱屑过程中发挥着重要作用。在本研究中，我们检测了重构表皮中的钙分布情况，以试图了解体外角质形成细胞分化和脱屑的生理学机制。采用离子捕获细胞化学方法（草酸钾-焦锑酸钾法）来定位在三种不同培养条件下（含血清培养基、无血清培养基以及添加视黄酸的无血清培养基）生成的重构表皮中的离子钙，从而比较不完全分化和充分分化角质形成细胞的生理学特性。在含血清培养基中生成的重构表皮呈现出不完全分化的特征，与天然皮肤相比，角质层中不完全分化细胞内的钙含量较高。使用含有维生素和脂质补充剂的无血清培养基可显著改善角质层超微结构以及穿过角质层的渗透途径，表明重构表皮的屏障形成得到改善。与此同时，钙分布模式正常化，显示颗粒层中的钙含量最高，而角质层内层中的钙含量较低。向无血清培养基中添加视黄酸会导致角质形成细胞分化改变，并在角质层中再次出现大量钙沉淀。应用质子探针X射线微分析法对无血清培养基中生成的天然和重构表皮中的钙分布进行定量研究，并验证了沉淀技术所显示的钙分布情况。无论角质层中是否存在钙，所有检测的培养系统均表现出脱屑不足，这与角质层糜蛋白酶主要以无活性前体形式存在的发现相关。本研究表明，体外角质层屏障特性的改善与表皮钙梯度的正常化同时发生，而终末分化失调与不完全分化的角质形成细胞内钙离子的积累相关。
