Shiseido Life Science Research Center, Tsuzuki-ku, Yokohama 224-8558, Japan.
Int J Cosmet Sci. 2003 Oct;25(5):245-57. doi: 10.1046/j.1467-2494.2003.00194.x.
The cornified envelope (CE) is a thin insoluble structure enveloping corneocytes, and is essential for the barrier function of the stratum corneum (SC). Our previous studies revealed that immature CEs are detected in the outermost layer of SC of barrier-impaired epidermis including the face and in various inflammatory disorders, using a non-invasive method to evaluate CE maturity. However, factors attributable for immaturity of CEs are still unclear. The aim of the present study is to clarify whether immature CEs in the SC have the potential to mature. SC samples, in which immature CEs abundantly exist, were collected from the cheek of healthy volunteers by tape-stripping, and were incubated ex vivo under the humidified air at 37 degrees C. Then, CE maturity was evaluated by staining with a combination of anti-involucrin and Nile red to detect involucrin antigenicity in the immature CEs and hydrophobicity in the mature CEs, respectively. Ex vivo incubation of the SC resulted in the conversion of immature CEs into mature CEs in terms of loss of involucrin antigenicity and acquisition of hydrophobicity. Application of buffer solutions of various pH onto the SC prior to incubation revealed that maturation of CEs was proceeded at range of pH 5-7, corresponding to intrinsic pH range within the SC. Chelating agents, ethylenediamine-N, N, N', N',-tetraacetic acid (EDTA) and ethyleneglycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and thiol alkylating agents, N-ethylmaleimide and iodoacetamide, inhibited the maturation. Labelled cadaverine as an exogenous substrate for transglutaminase (TGase) could be incorporated into CEs during maturation. Extractable involucrin-like protein detected in the SC samples before incubation concomitantly disappeared with CE maturation, suggesting incorporation of endogenous substrates into the CEs. These results obviously demonstrate that maturation of CEs was mediated by TGase activity in the SC, and that immature CEs found in the outermost face SC have potential to mature by cross-linking of endogenous CE precursors present in the SC. Reduction of environmental humidity during ex vivo incubation of the SC resulted in marked suppression of maturation of CEs, and application of a moisturizer, glycerine, onto the SC replenished the suppression of maturation, suggesting that water content in the SC may affect the TGase reaction in the SC. Therefore, various factors, including a decrease in the water content in the SC, may account for impaired maturation of CEs in the face SC.
角蛋白包膜（CE）是包裹角质细胞的薄而不可溶的结构，对于角质层（SC）的屏障功能至关重要。我们之前的研究使用非侵入性方法评估 CE 成熟度，发现包括面部在内的屏障受损表皮和各种炎症性疾病的 SC 最外层存在不成熟的 CE。然而，导致 CE 不成熟的因素仍不清楚。本研究旨在阐明 SC 中不成熟的 CE 是否具有成熟的潜力。通过胶带剥离从健康志愿者的脸颊上采集 SC 样本，其中大量存在不成熟的 CE，并在 37°C 的加湿空气中进行体外孵育。然后，通过抗包裹蛋白和尼罗红的组合染色分别检测不成熟 CE 中的包裹蛋白抗原性和成熟 CE 的疏水性来评估 CE 成熟度。SC 的体外孵育导致不成熟 CE 转化为成熟 CE，表现为包裹蛋白抗原性丧失和获得疏水性。在孵育前将各种 pH 值的缓冲液应用于 SC 表明，CE 的成熟是在 pH5-7 的范围内进行的，这与 SC 内的固有 pH 范围相对应。螯合剂乙二胺四乙酸（EDTA）和乙二醇双（β-氨基乙基醚）-N，N，N'，N'-四乙酸（EGTA）和巯基烷化剂 N-乙基马来酰亚胺和碘乙酰胺抑制了成熟。作为转谷氨酰胺酶（TGase）的外源底物标记的腐胺可在成熟过程中掺入 CE。孵育前 SC 样本中可检测到的可提取包裹蛋白样蛋白与 CE 成熟同时消失，表明内源性底物掺入 CE。这些结果明显表明，SC 中的 TGase 活性介导了 CE 的成熟，并且在 SC 中发现的最外层面部 SC 中的不成熟 CE 具有通过交联 SC 中存在的内源性 CE 前体成熟的潜力。在 SC 的体外孵育过程中降低环境湿度会显著抑制 CE 的成熟，并且将保湿剂甘油应用于 SC 可补充对成熟的抑制作用，这表明 SC 中的水分含量可能会影响 SC 中的 TGase 反应。因此，包括 SC 中水分含量降低在内的各种因素可能导致面部 SC 中 CE 成熟受损。
