Garlick J A, Taichman L B
Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook.
Lab Invest. 1994 Jun;70(6):916-24.
Reepithelialization of an incisional wound in a stratified squamous epithelium is accomplished by mobilizing keratinocytes from the wound margins. In vitro models to study this phenomenon have been limited by incomplete differentiation of the cultured epithelium. In addition, it has been difficult to follow fate of recruited keratinocytes, since techniques for marking cells have not been available. We have adapted an organotypic culture model in which keratinocytes are fully differentiated and have utilized a genetic marking protocol with retroviral vectors to study reepithelialization after an incisional wound.
The fully differentiated epithelium of an organotypic culture model was incised, supported on a collagen matrix, and allowed to reepithelialize. At various times after wounding, healing cultures were monitored for migration, differentiation, and proliferation by immunohistochemical staining. Histochemical staining specific for the genetically marked cells assisted in the determination of how these cells behaved during reepithelialization.
The first event observed (at 8 hours) was migration of suprabasal keratinocytes into the wound followed by a transient proliferative burst at the wound margin. Reepithelialization was complete by 24 hours. Proliferation in the wound epithelium persisted during stratification and terminal differentiation. Genetically marked cells in the wound epithelium were present in clusters demonstrating that proliferation and displacement of cells occurred near the edge of the epithelial tongue. Individual genetically marked cells were also found in the wound epithelium, indicating that individual cells had migrated a considerable distance from the wound edge without having undergone replication.
This is the first report of an organotypic model for reepithelialization, and we demonstrate that migration, proliferation, and differentiation occur during this process. The proliferative response which follows initial cell migration at the wound margins suggests that these events are temporally coordinated as phenotypically different populations of cells are sequentially activated. By following the distribution of genetically marked cells in the wound, it is evident that at least two types of cells repopulate a wound-proliferative and migratory cells.
分层鳞状上皮切口创面的再上皮化是通过动员创面边缘的角质形成细胞来完成的。用于研究这一现象的体外模型受到培养上皮细胞不完全分化的限制。此外，由于缺乏标记细胞的技术，追踪募集的角质形成细胞的命运一直很困难。我们采用了一种角质形成细胞能完全分化的器官型培养模型，并利用逆转录病毒载体的基因标记方案来研究切口创面后的再上皮化过程。
在胶原基质上对器官型培养模型的完全分化上皮进行切割，并使其再上皮化。在创伤后的不同时间，通过免疫组织化学染色监测愈合培养物的迁移、分化和增殖。对基因标记细胞的组织化学染色有助于确定这些细胞在再上皮化过程中的行为。
观察到的第一个事件（在8小时时）是基底上层角质形成细胞迁移到创面，随后创面边缘出现短暂的增殖爆发。24小时时再上皮化完成。创面上皮的增殖在分层和终末分化过程中持续存在。创面上皮中的基因标记细胞呈簇状分布，表明细胞的增殖和移位发生在上皮舌边缘附近。在创面上皮中也发现了单个基因标记细胞，表明单个细胞从创面边缘迁移了相当远的距离而未进行复制。
这是关于再上皮化的器官型模型的首次报道，我们证明了在此过程中发生了迁移、增殖和分化。创面边缘初始细胞迁移后的增殖反应表明，随着表型不同的细胞群体被依次激活，这些事件在时间上是协调的。通过追踪创面中基因标记细胞的分布，很明显至少有两种类型的细胞重新填充创面——增殖细胞和迁移细胞。
