Application of a partial-thickness human ex vivo skin culture model in cutaneous wound healing study.

A number of in vivo and ex vivo skin models have been applied to human wound healing studies. A reliable skin model, which recapitulates the features of human wound repair, is essential for the clinical and mechanical investigation of human cutaneous wound healing. Full-skin ex vivo culture systems have been used in wound healing studies. However, important structures of the skin, such as the differentiation of keratinocytes and epidermis-dermis junction, are poorly characterized in this model. This study aims to develop an optimized partial-thickness human ex vivo skin culture (HESC) model to maintain human skin characteristics in vitro. During our culture, the basal layer, suprabasal layer, and stratum granulosum layer of epidermis were preserved until day 8. Analyses of hemidesmosome proteins, bullous pemphigoid antigen 1 (BP180) and 2 (BP230), showed that the integrity of the basement membrane of the epidermis was well preserved in the HESC model. In contrast, an organotypic culture with human keratinocytes and fibroblasts failed to show an integrated basement membrane. Maintenance of skin structure by histological analysis and proliferation of epidermal keratinocytes by Ki67 staining were observed in our model for 12 days. Complete re-epithelialization of the wounding area was observed at day 6 post wounding when a superficial incisional wound was created. The expression of Ki-67 and keratin 6, indicators of activated keratinocytes in epidermis, was significantly upregulated and new collagen synthesis was found in the dermis during the wound healing process. As control, we also used organotypic culture in studying the differentiation of the keratinocyte layers and incisional wound repair. It turned out that our model has advantage in these study fields. The results suggest that our HESC model retains important elements of in vivo skin and has significant advantages for the wound healing studies in vitro.

Molecular cloning and characterization of the promoter region of the human Phox2b gene.

The closely related homeodomain transcription factors, Phox2a and Phox2b, are restrictively expressed in central and peripheral noradrenergic (NA) neurons in an overlapping but distinct manner, and critically regulate the differentiation and neurotransmitter identity of NA neurons. The structure and function of the human Phox2a (hPhox2a) promoter has recently been reported. Towards the long-term goal of delineating the regulatory cascade of NA neuron differentiation, we isolated a human Phox2b (hPhox2b) genomic clone encompassing approximately 7.8 kb of the 5' upstream promoter region, the entire exon-intron structure and 4.5 kb of the 3' flanking region. Two transcription start sites are identified to reside 115 and 110 nucleotides upstream of the start codon, based on both primer extension and 5'-rapid amplification of the cDNA ends analyses. In addition, transient transfection assays indicate that 1.1 kb or longer upstream sequences of the hPhox2b gene may confer cell type-specific gene expression in certain, but not all cell lines. The promoter activity of the hPhox2b gene is modestly transactivated by forced co-expression of Phox2b and the hPhox2b gene promoter contains a high-affinity binding site at -320 to -295 bp. This study provides a frame to further elucidate the molecular mechanisms underlying the regulation of Phox2a and Phox2b gene expression and its relation to NA differentiation.