Alitalo K, Kuismanen E, Myllylä R, Kiistala U, Asko-Seljavaara S, Vaheri A
J Cell Biol. 1982 Sep;94(3):497-505. doi: 10.1083/jcb.94.3.497.
Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.
使用经辐照的BALB/3T3细胞作为饲养层，启动从成人表皮获取的人表皮角质形成细胞的培养。在表皮岛融合的不同阶段，去除饲养细胞，并通过生化和免疫化学方法分析纯组分细胞和共培养物的细胞外基质蛋白，并与相同供体的皮肤成纤维细胞的蛋白进行比较。角质形成细胞合成并分泌纤连蛋白以及少量层粘连蛋白和IV型胶原。此外，角质形成细胞合成了一种非二硫键连接的胶原多肽（Mr = 120,000），且局限于细胞层。表皮组织的器官培养物也合成了Mr = 120,000的胶原多肽，并在其酸性或去污剂提取物中检测到，但同样未发现其分泌到培养基中。Mr = 120,000的胶原具有与I - V型胶原不同的生化和免疫学特性。在角质形成细胞培养物的免疫荧光中，纤连蛋白染色在表皮细胞岛扩展周边的边缘衬里细胞中很突出，但在分层集落上层的终末分化细胞中未检测到。发现角质形成细胞以细胞周基质形式沉积的IV型胶原非常少。相比之下，发现小鼠3T3饲养细胞除了先前鉴定的成纤维细胞结缔组织糖蛋白、间质前胶原和纤连蛋白外，还产生IV型胶原和层粘连蛋白。发现3T3细胞的基底膜胶原以明显未加工的前胶原α1(IV)和α2(IV)链的形式沉积。成纤维细胞饲养细胞在培养条件下产生基底膜糖蛋白可能促进共培养的角质形成细胞的附着和生长。
