Glasø M, Håskjold E
Electron Microscopical Laboratory, University of Oslo, Norway.
Virchows Arch B Cell Pathol Incl Mol Pathol. 1989;57(3):181-94. doi: 10.1007/BF02899080.
The denuded basal cell layer of the hairless mouse epidermis is described in the present scanning (SEM) and transmission electron microscopical (TEM) study. The suprabasal layers were removed mechanically after trypsinization or by extracellular calcium depletion. Trypsinization before removal of the suprabasal cells caused the basal cells to shrink. Characteristic surface plication and hemi-desmosomal attachment to the basement membrane were generally preserved. SEM revealed partly maintained intercellular bridging, whereas by TEM such contacts were absent because half desmosomes were internalized. Total calcium depletion induced more serious damage to the basal cell surface, which was smooth with apparent perforations. However, cell bridges, and occasional desmosomes were present. The cell interior demonstrated important cellular injury. If the calcium deprived explants were allowed to recover in calcium-containing medium, the cells acquired an activated "regenerative" morphology, without junctions, similar to that observed in wound healing. Epidermal non-keratinocytes were seen only after trypsinization. Control experiments revealed that they adapted poorly to organ culture conditions. By TEM, we observed several interesting aspects of the differences, between dark and clear basal keratinocytes. This was unexpected because fixation studies had shown, that with the present fixation method, typical dark and clear cells do not occur in untreated epidermis. We believe that membrane injury through mechanical stripping of partly adhering epidermal layers induced "clear cells", whereby the neighboring cells appeared darker. This provides additional evidence as to the origin of the two sub-populations, dark and clear basal cells. The clear cells may be injured cells, caused by cell damage, and not by processes of cellular differentiation. The results of the present investigation supports the view that basal keratinocytes have a polygonal shape with numerous free surface extensions and they are anchored to the basement membrane with "foot pads". Our study also shows that SEM of the epidermal basal layer might be feasible. Various artifacts, however, must be considered, depending on the denudation method used. We prefer trypsinization to calcium depletion because it is less time-consuming and results in a cell morphology which in TEM is comparable to that of basal cells in untreated whole epidermis. Extra-cellular calcium depletion, however, might be useful as a method to prepare single cell suspensions for flow cytometry. Restoration of a normal calcium concentration after stripping, provides an opportunity to mimic wound healing in situ, as an alternative t
在本扫描电子显微镜（SEM）和透射电子显微镜（TEM）研究中，对无毛小鼠表皮裸露的基底细胞层进行了描述。在胰蛋白酶消化后或通过细胞外钙耗尽，机械去除基底上层。在去除基底上层细胞之前进行胰蛋白酶消化会导致基底细胞收缩。特征性的表面褶皱和与基底膜的半桥粒附着通常得以保留。SEM显示部分细胞间桥得以保留，而通过TEM观察，由于半桥粒内化，此类接触并不存在。完全钙耗尽对基底细胞表面造成更严重的损伤，表面光滑且有明显穿孔。然而，细胞桥和偶尔的桥粒存在。细胞内部显示出重要的细胞损伤。如果将缺钙的外植体置于含钙培养基中恢复，细胞会呈现出活化的“再生”形态，没有细胞连接，类似于伤口愈合时观察到的形态。仅在胰蛋白酶消化后可见表皮非角质形成细胞。对照实验表明它们对器官培养条件适应较差。通过TEM，我们观察到了暗基底角质形成细胞和亮基底角质形成细胞之间差异的几个有趣方面。这是出乎意料的，因为固定研究表明，使用当前的固定方法，在未处理的表皮中不会出现典型的暗细胞和亮细胞。我们认为，通过机械剥离部分附着的表皮层造成的膜损伤诱导产生了“亮细胞”，从而使相邻细胞显得更暗。这为暗基底细胞和亮基底细胞这两个亚群的起源提供了额外证据。亮细胞可能是由细胞损伤而非细胞分化过程导致的受损细胞。本研究结果支持这样的观点，即基底角质形成细胞呈多边形，有许多自由表面延伸，并且通过“脚垫”锚定在基底膜上。我们的研究还表明，对表皮基底层进行SEM观察可能是可行的。然而，根据所使用的剥脱方法，必须考虑各种假象。我们更倾向于胰蛋白酶消化而非钙耗尽，因为它耗时更少，并且在TEM下产生的细胞形态与未处理的全表皮中的基底细胞形态相当。然而，细胞外钙耗尽作为一种制备用于流式细胞术的单细胞悬液的方法可能是有用的。剥脱后恢复正常钙浓度，为原位模拟伤口愈合提供了机会，作为一种替代方法……
