Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.
School of Biomedical Sciences, Faculty of Health, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.
Br J Dermatol. 2017 Jan;176(1):145-158. doi: 10.1111/bjd.14879. Epub 2016 Dec 22.
Epidermal homeostasis is maintained through the balance between keratinocyte proliferation, differentiation and desquamation; however, human skin equivalent (HSE) models are known to differentiate excessively. In native tissue, proteases such as kallikrein-related peptidase (KLK) 5 and KLK7 cleave the extracellular components of corneodesmosomes; proteins corneodesmosin, desmocollin 1 and desmoglein 1, loosening the cellular connections and enabling desquamation. The actions of KLK7 are tightly controlled by protease inhibitors, skin-derived antileucoproteinase (SKALP) and lymphoepithelial Kazal-type-related inhibitor (LEKTI), which also inhibits KLK5, localizing protease activity to the stratum corneum.
To investigate the mechanisms that inhibit the desquamation cascade in HSE models.
Human skin tissue and HSE models were investigated using gene microarray, real-time polymerase chain reaction (PCR), immunohistochemistry and Western blot analysis to examine key components of the desquamation pathway. To elucidate proteolytic activity in HSEs and native skin, in situ and gel zymography was performed.
Histological analysis indicated that HSE models form a well-organized epidermis, yet develop an excessively thick and compact stratum corneum. Gene microarray analysis revealed that the desquamation cascade was dysregulated in HSE models and this was confirmed using real-time PCR and immunohistochemistry. Immunohistochemistry and Western blot indicated overexpression of LEKTI and SKALP in HSEs. Although KLK7 was also highly expressed in HSEs, zymography indicated that protease activation and activity was lower than in native skin.
These findings demonstrate that stratum corneum thickening is due to inhibited KLK5 and KLK7 activation and a subsequent lack of corneodesmosome degradation in the HSE model epidermis.
表皮的稳态通过角质形成细胞的增殖、分化和脱屑之间的平衡来维持；然而，众所周知，人体皮肤等效物（HSE）模型过度分化。在天然组织中，丝氨酸蛋白酶如 Kallikrein-related peptidase (KLK) 5 和 KLK7 切割桥粒芯糖蛋白的细胞外成分；桥粒芯糖蛋白、桥粒斑珠蛋白 1 和桥粒芯胶蛋白 1 等蛋白使细胞连接变松，从而促进脱屑。KLK7 的作用受到丝氨酸蛋白酶抑制剂、皮肤衍生的抗白细胞蛋白酶（SKALP）和淋巴上皮 Kazal 型相关抑制剂（LEKTI）的严格控制，后者还抑制 KLK5，将蛋白酶活性定位在角质层。
研究抑制 HSE 模型脱屑级联的机制。
通过基因微阵列、实时聚合酶链反应（PCR）、免疫组织化学和 Western blot 分析研究人皮肤组织和 HSE 模型，以检查脱屑途径的关键成分。为了阐明 HSE 和天然皮肤中的蛋白水解活性，进行了原位和凝胶酶谱分析。
组织学分析表明，HSE 模型形成了一个组织良好的表皮，但形成了一个异常厚且致密的角质层。基因微阵列分析表明，HSE 模型中的脱屑级联失调，这通过实时 PCR 和免疫组织化学得到了证实。免疫组织化学和 Western blot 表明 HSE 中 LEKTI 和 SKALP 的过度表达。尽管 KLK7 在 HSE 中也高度表达，但酶谱分析表明蛋白酶的激活和活性低于天然皮肤。
这些发现表明，角质层增厚是由于 HSE 模型表皮中 KLK5 和 KLK7 激活受到抑制，以及随后桥粒芯糖蛋白降解的缺乏所致。
