Boderke P, Boddé H E, Ponec M, Wolf M, Merkle H P
Department of Pharmacy, Swiss Federal Institute of Technology Zurich, ETH, Switzerland.
J Investig Dermatol Symp Proc. 1998 Aug;3(2):180-4. doi: 10.1038/jidsymp.1998.35.
HaCaT cell culture sheets were recently demonstrated to be a useful tool to study epidermal metabolism. Here we report on a mechanistic and quantitative correlation between the kinetics of aminopeptidase-based cleavage of L-Ala-4-methoxy-2-naphthylamide (Ala-MNA) in HaCaT sheets versus stripped human skin. Fresh human skin (breast or abdominal) was obtained from cosmetic surgery, tape-stripped, and dermatomed. HaCaT sheets were cultured on porous membranes. Diffusion and concurrent metabolism were studied under reflection and permeation conditions. Numerical simulations of simultaneous diffusion and saturable Michaelis-Menten metabolism were based on a physical model and a fixed set of independently obtained parameters (diffusion coefficient D, distance x, partition coefficient P, Michaelis constant Km, maximum metabolic rate Vmax). Under reflection conditions, cleavage of Ala-MNA in HaCaT sheets was very close to stripped skin. In contrast, in permeation studies substrate only permeated through HaCaT whereas passage through stripped skin led to full cleavage of Ala-MNA to MNA. All experimental data were in reasonable to excellent agreement with numerically generated data. Differences between HaCaT and stripped skin could be quantitatively and mechanistically explained by the thickness of the metabolically active layer, i.e., approximately 10 microm in HaCaT and approximately 40 microm in stripped skin. Full cleavage of permeating Ala-MNA in stripped skin was predicted to occur within the upper approximately 20 microm of viable epidermis. Thus epidermal aminopeptidase activity may act as an efficient metabolic barrier to fully block the permeation of aminopeptidase labile xenobiotics. Within the settings of this study the kinetics of metabolism in the viable epidermis of skin is predictable from HaCaT sheets.
最近有研究表明，HaCaT细胞培养片是研究表皮代谢的一种有用工具。在此，我们报告了HaCaT细胞培养片与去除角质的人体皮肤中基于氨肽酶对L-丙氨酸-4-甲氧基-2-萘酰胺（Ala-MNA）的裂解动力学之间的机制和定量相关性。新鲜人体皮肤（乳房或腹部）取自整形手术，经胶带剥离和削薄处理。将HaCaT细胞培养片培养在多孔膜上。在反射和渗透条件下研究了扩散和同时进行的代谢。基于物理模型和一组独立获得的固定参数（扩散系数D、距离x、分配系数P、米氏常数Km、最大代谢速率Vmax），对同时扩散和饱和米氏代谢进行了数值模拟。在反射条件下，HaCaT细胞培养片中Ala-MNA的裂解与去除角质的皮肤非常接近。相比之下，在渗透研究中，底物仅透过HaCaT细胞培养片，而透过去除角质的皮肤会导致Ala-MNA完全裂解为MNA。所有实验数据与数值生成的数据具有合理到极好的一致性。HaCaT细胞培养片与去除角质的皮肤之间的差异可以通过代谢活性层的厚度进行定量和机理解释，即HaCaT细胞培养片中约为10微米，去除角质的皮肤中约为40微米。预计去除角质的皮肤中渗透的Ala-MNA的完全裂解发生在活表皮上部约20微米范围内。因此，表皮氨肽酶活性可能作为一种有效的代谢屏障，完全阻止氨肽酶不稳定的外源性物质的渗透。在本研究的条件下，皮肤活表皮中的代谢动力学可从HaCaT细胞培养片中预测。
