Yang Lujun, Shirakata Yuji, Shudou Masachika, Dai Xiuju, Tokumaru Sho, Hirakawa Satoshi, Sayama Koji, Hamuro Junji, Hashimoto Koji
Department of Dermatology, Ehime University School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan.
Cell Tissue Res. 2006 Oct;326(1):69-77. doi: 10.1007/s00441-006-0208-2. Epub 2006 Jun 7.
The presence of pre-existing basement membrane (BM) components improves the morphogenesis of epidermis and BM in constructing a human living skin-equivalent (LSE). De-epithelialized amniotic membrane (AM) retains key BM components. We have therefore investigated the usefulness of AM for constructing LSE. De-epithelialized AM was overlaid on type I collagen gel embedded with fibroblasts. Normal human keratinocytes (NHKs) were then seeded onto the epithelial side of the AM to construct an AM-LSE. A conventional LSE was constructed by seeding NHKs on a fibroblast-populated type I collagen gel. When the keratinocytes reached confluence, the LSE was lifted to the air-liquid interface and cultured for up to 3 weeks. Samples were harvested at various times and investigated morphologically, immunohistochemically, and ultrastructurally. In AM-LSE, the epidermis was better stratified, with more compact, polarized, columnar basal cells, and the expression of differentiation and proliferation markers was more similar to that of normal human skin than was that of LSE without AM. A more continuous BM and better-developed hemidesmosomes were found in AM-LSE. The epidermis of AM-LSE outgrew much faster than that of LSE without AM. When transplanted onto nude mice, both LSEs took well; however, the AM-LSE graft showed better morphogenesis of the epidermis, BM, and hemidesmosomes. The better epidermal morphology and better-developed BM in AM-LSE in vitro and in vivo indicates its superiority over LSE without AM for clinical applications.
预先存在的基底膜（BM）成分的存在，在构建人活皮肤替代物（LSE）时可改善表皮和BM的形态发生。去上皮羊膜（AM）保留了关键的BM成分。因此，我们研究了AM在构建LSE中的作用。将去上皮的AM覆盖在包埋有成纤维细胞的I型胶原凝胶上。然后将正常人角质形成细胞（NHK）接种到AM的上皮侧以构建AM-LSE。通过将NHK接种在成纤维细胞填充的I型胶原凝胶上来构建传统的LSE。当角质形成细胞达到汇合状态时，将LSE提升至气液界面并培养长达3周。在不同时间收集样本，并进行形态学、免疫组织化学和超微结构研究。在AM-LSE中，表皮分层更好，基底细胞更致密、极化且呈柱状，与不含AM的LSE相比，分化和增殖标志物的表达与正常人皮肤更相似。在AM-LSE中发现了更连续的BM和发育更好的半桥粒。AM-LSE的表皮生长速度比不含AM的LSE快得多。当移植到裸鼠身上时，两种LSE都生长良好；然而，AM-LSE移植物的表皮、BM和半桥粒的形态发生更好。AM-LSE在体外和体内具有更好的表皮形态和发育更好的BM，表明其在临床应用中优于不含AM的LSE。
