A Human Epidermal Model Grown On An Acellular Gel
JOURNAL OF TOXICOLOGY-CUTANEOUS AND OCULAR TOXICOLOGY(1993)
摘要
An in vitro epidermis was constructed from normal human epidermal keratinocytes (NHEK) grown on a cross-linked, acellular, type I collagen gel. The latter was overlaid on a Millipore Millicell CM microporous membrane. This human epidermal model (HEM) was differentiated in the presence of elevated levels of calcium and raised to the air-liquid interface. The HEM differentiated into 20+ cell layers and demonstrated ultrastructural characteristics of human epidermis in vivo, including an electrondense stratum corneum, keratin filaments, desmosomes, lamellar-like bodies, and, most notably, a structure resembling the basal lamina at the interface of the epidermis and the gel. This basal lamina appeared in preparations grown at either the air-liquid interface or under more conventional, submerged conditions. A planar basal surface was a corequisite for the appearance of the basal lamina in both cases. Because this HEM is one of the few in vitro epidermal models reported to construct a structure resembling the basal lamina, we asked if the cross-linked collagen gel substrate might be influencing the synthesis and/or secretion of particular extracellular matrix components, which, in turn, might result in the assembly of a basal lamina. To this end, laminin synthesis and secretion were analyzed in response to variations in the matrix form on which the HEM differentiated. More beta-laminin was synthesized when keratinocytes were plated on type I collagen gel (CG) substrates compared to a dry type I collagen film (DCF). This apparent increase in synthesis occurred whether a microporous membrane or a conventional plastic surface was used as the cell culture device. More beta laminin was secreted apically by HEM on DCF-coated surfaces than on CG-coated surfaces; this appeared to be independent of the cell culture device used. Less basal beta laminin, however, was secreted by HEM on DCF-coated Millicell CM microporous membranes than CG-coated microporous membranes. Thus it appears that the CG increases beta laminin synthesis and alters beta laminin trafficking patterns so that more of this extracellular matrix component is secreted basally rather than apically. This implies that the matrix form of type I collagen can direct the synthesis and secretion of laminin. Consideration of the proper matrix forms, as well as its molecular constituency, might be a key factor in regulating the deposition of a basal lamina by reconstructed human epidermis in vitro.
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关键词
human epidermal model grown,acellular gel
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