Woodley David T, Krueger Gerald G, Jorgensen Cynthia M, Fairley Janet A, Atha Tom, Huang Yi, Chan Lawrence, Keene Douglas R, Chen Mei
Department of Medicine, Division of Dermatology, University of Southern California, Los Angeles, California 90033, USA.
J Invest Dermatol. 2003 Nov;121(5):1021-8. doi: 10.1046/j.1523-1747.2003.12571.x.
Type VII collagen is synthesized and secreted by both human keratinocytes and fibroblasts. Although both cell types can secrete type VII collagen, it is thought that keratinocytes account for type VII collagen at the dermal-epidermal junction (DEJ). In this study, we examined if type VII collagen secreted solely by dermal fibroblasts could be transported to the DEJ. We established organotypic, skin-equivalent cultures composed of keratinocytes from patients with recessive dystrophic epidermolysis bullosa (RDEB) and normal dermal fibroblasts. Immuno-labeling of skin equivalent sections with the anti-type VII collagen antibody revealed tight linear staining at the DEJ. RDEB fibroblasts, were gene-corrected to make type VII collagen and used to regenerate human skin on immune-deficient mice. The human skin generated by gene-corrected RDEB fibroblasts or normal human fibroblasts combined with RDEB keratinocytes restored type VII collagen expression at the DEJ in vivo. Further, intradermal injection of normal human or gene-corrected RDEB fibroblasts into mouse skin resulted in the stable expression of human type VII collagen at the mouse DEJ. These data demonstrate that human dermal fibroblasts alone are capable of producing type VII collagen at the DEJ, and it is possible to restore type VII collagen gene expression in RDEB skin in vivo by direct intradermal injection of fibroblasts.
VII型胶原蛋白由人类角质形成细胞和成纤维细胞合成并分泌。虽然这两种细胞类型都能分泌VII型胶原蛋白，但一般认为在真皮-表皮交界处（DEJ）的VII型胶原蛋白主要由角质形成细胞产生。在本研究中，我们检测了仅由真皮成纤维细胞分泌的VII型胶原蛋白是否能够转运至DEJ。我们建立了由隐性营养不良性大疱性表皮松解症（RDEB）患者的角质形成细胞和正常真皮成纤维细胞组成的器官型皮肤等效物培养体系。用抗VII型胶原蛋白抗体对皮肤等效物切片进行免疫标记，结果显示在DEJ处有紧密的线性染色。将RDEB成纤维细胞进行基因校正以使其能够产生VII型胶原蛋白，并用于在免疫缺陷小鼠身上再生人类皮肤。由基因校正的RDEB成纤维细胞或正常人类成纤维细胞与RDEB角质形成细胞联合产生的人类皮肤在体内恢复了DEJ处VII型胶原蛋白的表达。此外，将正常人类或基因校正的RDEB成纤维细胞皮内注射到小鼠皮肤中，可使人类VII型胶原蛋白在小鼠DEJ处稳定表达。这些数据表明，仅人类真皮成纤维细胞就能够在DEJ处产生VII型胶原蛋白，并且通过直接皮内注射成纤维细胞有可能在体内恢复RDEB皮肤中VII型胶原蛋白基因的表达。
