Boderke P, Merkle H P, Cullander C, Ponec M, Boddé H E
Department of Pharmacy, Swiss Federal Institute of Technology Zurich (ETH), Switzerland.
J Invest Dermatol. 1997 Jan;108(1):83-6. doi: 10.1111/1523-1747.ep12285642.
The aim of this study was to localize and visualize aminopeptidase activity within freshly excised, dermatomed human skin without perturbation of its histologic integrity. The use of confocal laser scanning microscopy (CLSM) is introduced as a novel approach by which to monitor the degradation of suitable substrates in the skin. The fluorescence of the metabolites originating from the cleavage of the aminopeptidase probe bis-Leu-rhodamine 110 (Leu2-R11O) was interpreted to reflect the local aminopeptidase activity in the tissue. To separate the kinetics of diffusion and degradation of Leu2-R110, a lateral application mode was introduced: the probe was applied at the cutting plane of a mechanical cross-section of the sample, and optical cross-sections were made parallel to the cutting plane of the mechanical section. By this means, simultaneous and equal access of the substrate to the various strata and domains of the skin was achieved. The observations revealed that the fluorescence, i.e., aminopeptidase activity, was evenly distributed throughout the viable part of the epidermis, with enhanced fluorescence ("hot spots") in the upper layers of the stratum granulosum, while dermis and stratum corneum showed considerably less aminopeptidase activity. Independent studies with hair follicles (obtained from trypsin-separated stratum corneum) also showed aminopeptidase activity, mostly at the root sheath. Because of the advantage of direct visualization and localization of enzymatic activity in intact tissue, the lateral application mode of substrate administration in combination with CLSM may be beneficial to further elucidate the location and intensity of metabolic activity in other living tissues as well.
本研究的目的是在不破坏其组织学完整性的情况下，对新鲜切除的、经皮片移植的人皮肤中的氨肽酶活性进行定位和可视化。引入共聚焦激光扫描显微镜（CLSM）作为一种新方法，用于监测皮肤中合适底物的降解。源自氨肽酶探针双亮氨酸罗丹明110（Leu2-R11O）裂解的代谢物荧光被解释为反映组织中的局部氨肽酶活性。为了区分Leu2-R110的扩散和降解动力学，引入了一种侧向应用模式：将探针应用于样品机械横截面的切割平面，并制作与机械截面切割平面平行的光学截面。通过这种方式，实现了底物同时且平等地进入皮肤的各个层和区域。观察结果显示，荧光，即氨肽酶活性，在表皮的存活部分均匀分布，在颗粒层上层荧光增强（“热点”），而真皮和角质层的氨肽酶活性则明显较低。对毛囊（从胰蛋白酶分离的角质层中获得）的独立研究也显示了氨肽酶活性，主要在毛根鞘处。由于在完整组织中直接可视化和定位酶活性的优势，底物给药的侧向应用模式与CLSM相结合可能也有利于进一步阐明其他活组织中代谢活性的位置和强度。
