Germain L, Guignard R, Rouabhia M, Auger F A
Laboratoire des Grands Brûlés/LOEX, Hôpital du Saint-Sacrement, Sainte-Foy, Québec, Canada.
Burns. 1995 May;21(3):175-80. doi: 10.1016/0305-4179(95)80004-8.
The basement membrane zone is important for graft adhesion and stability. The aim of the present study was to visualize the regeneration of the basement membrane and determine the sequential appearance of its constituents in the early postgrafting period of cultured human epidermal sheets. A keratinocyte single cell suspension, devoid of dermal fibroblast contamination, was obtained from human skin by a two-step tissue digestion method with thermolysin and trypsin. After culturing, epidermal sheets were generated, detached enzymatically by incubating with thermolysin (for 20-30 min) or Dispase (for 45-60 min), and deposited on a muscular graft bed of athymic mice. Immunohistochemistry and ultrastructural analyses were performed on biopsies harvested 2, 4 and 21 days postgrafting. Bullous pemphigoid antigens and laminin were detected at the dermo-epidermal junction, showing an almost continuous line 2 days postgrafting. Type IV collagen was generally absent at this time, but it was detected 4 days postgrafting. Type VII collagen was labelled as a discontinuous line of increasing intensity from 2 to 21 days postgrafting. Ultrastructural analysis revealed hemidesmosomes and a discontinuous lamina densa 2 days postgrafting, and a complete basement membrane with a continuous lamina densa, hemidesmosomes and anchoring fibrils 21 days postgrafting. The sequence of appearance of major basement membrane components was similar for cultured sheets detached with thermolysin or Dispase. However, it differed from that of other wound healing models. Results are discussed in terms of the variable keratinocyte migration requirement between various wound healing models.
基底膜区对于移植物的黏附与稳定性至关重要。本研究的目的是观察基底膜的再生情况，并确定其成分在培养的人表皮片移植后早期的相继出现情况。通过用嗜热菌蛋白酶和胰蛋白酶进行两步组织消化法从人皮肤中获得无真皮成纤维细胞污染的角质形成细胞单细胞悬液。培养后，生成表皮片，通过用嗜热菌蛋白酶（孵育20 - 30分钟）或中性蛋白酶（孵育45 - 60分钟）进行酶解分离，然后置于无胸腺小鼠的肌肉移植床上。在移植后2天、4天和21天采集活检组织进行免疫组织化学和超微结构分析。在真皮 - 表皮交界处检测到大疱性类天疱疮抗原和层粘连蛋白，移植后2天显示出几乎连续的线条。此时IV型胶原通常不存在，但在移植后4天可检测到。VII型胶原在移植后2天至21天被标记为强度增加的不连续线条。超微结构分析显示移植后2天有半桥粒和不连续的致密层，移植后21天有完整的基底膜，包括连续的致密层、半桥粒和锚定原纤维。用嗜热菌蛋白酶或中性蛋白酶分离的培养片主要基底膜成分的出现顺序相似。然而，它与其他伤口愈合模型不同。根据各种伤口愈合模型之间角质形成细胞迁移需求的差异对结果进行了讨论。
