Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions. Several methods to separate the dermis from the epidermis are available. The proteolytic enzyme thermolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation. In the present study an optimized cell isolation procedure using thermolysin and trypsin is described, which is particularly suitable for punch biopsies. A 16-20-h (overnight) incubation of biopsies taken from normal and hyperproliferative skin with thermolysin (0.5 mg/ml) at 4 degrees C produced a selective separation of the dermis and epidermis. After a 30-min trypsin incubation (0.25 mg/ml) at 37 degrees C a cell suspension was produced which was characterized by minimal cell damage (cellular debris and clumps), a high recovery of basal cells and high quality DNA histograms. Furthermore, dermal contamination was very low. The thermolysin-trypsin separation methodology followed by triple-labelling flow cytometry provided a precise quantification of the percentage of keratin 10-positive cells, vimentin-positive cells and cells in S and G2M phases. Proliferative activity was selectively measured in the basal, the suprabasal and the non-keratinocyte compartment at various time intervals during epidermal regeneration after adhesive tape stripping. In contrast to the non-keratinocytes, the percentage of cells in S and G2M phases in the basal keratinocytes and in the suprabasal compartment increased 44-48 h after stripping. The increased proliferation following tape stripping was paralleled by an increased invasion of vimentin-positive cells into the epidermis and preceded by a decreased number of keratin 10-positive cells. Thermolysin-trypsin separation followed by three-colour flow cytometry permits a highly selective characterization of normal and hyperproliferative epidermis.
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PMID:Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol. 896 93

We designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.
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PMID:Characterization of a new tissue-engineered human skin equivalent with hair. 1047 18

A cell-based wound coverage with keratinocytes and fibroblasts on the basis of a commercially available dermal substitute (Matriderm ((R)), Kollagen/Elastin matrix) was generated, in order to treat wide burn wounds. First the expansion of keratinocytes was optimised and the culturing time was minimised. Raw material was 1-2 cm (2) split skin. Dermis and epidermis were separated by enzymatic treatment with thermolysin. After treatment of both compartments with trypsin and collagenase I, keratinocytes and fibroblasts were isolated and expanded in collagen I coated dishes. After 10 days fibroblasts were seeded on Matriderm ((R)). After cultivation of the fibroblasts-containing matrix for one week keratinocytes were seeded on top. After an additional week of submersed cultivation the matrix was lifted up to the air-liquid interface to initiate epidermal cell differentiation. After 16 days in the air-liquid interphase the matrix was fixed and underwent immunohistochemical and electron microscopic analysis. Histological analysis showed a regularly stratification of the epidermal part. We observed collagen IV, a marker for the basement membrane, between epidermis and dermis. Desmoglein and the differentiation markers involucrine and cytokeratin 10 were found in the suprabasal layers of the epidermis. Electron microscopic analysis showed the basement membrane in the epidermal junction zone as well as cell-cell connections in the form of desmosomes. Late differentiation characteristics, like granular structures and the cornified layer, were found in the stratum granulosum and stratum corneum. Our results demonstrate that a skin equivalent can be generated by using a collagen/elastin matrix, with an expansion rate of 50-100-fold. This skin equivalent may be useful for covering deep wounds.
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PMID:[Development of an engraftable skin equivalent based on matriderm with human keratinocytes and fibroblasts]. 1971 Dec 56