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Basal Cell Carcimona (BCC) is the most common form of skin cancer in the UK, occurring most frequently on sun exposed sites such as the head, neck and trunk and has been associated with mutations in genes expressed by keratinocytes. In particular, the mutation of the Patched receptor of the Hedgehog signalling pathway results in the up regulation of the target transcription factor GU 1. BCCs also have a complex stroma which consists of dermal fibroblasts. Dermal fibroblasts form a supportive structure through the production and release of collagens for extracellular matrices and the expression of growth factors supportive to other localised cells types. Tumour derived fibroblasts have been shown to influence the growth of other forms of epithelial cancer such as breast and prostate, conferring tumourgenic properties and effecting normal growth. However, the properties of BCCs are poorly described and this is due in part to the difficulty in culturing BCCs. The aim of this study was to develop a method for successful extraction and culture of both BCC derived keratinocytes and dermal fibroblast, characterise and study any potential stromal effects of these cells. Keratinocyte and dermal fibroblast extractions were performed on 4 mm punch biopsies. BCC derived dermal fibroblasts were grown in comparison with normal human dermal fibroblasts extracted from normal human facelift skin. A Comparison of growth between normal dermal fibroblasts and BCC derived dermal fibroblasts showed BCC derived fibroblasts senescing under normal culture conditions. Immunocytochemistry detection of Beta Galactosidase was used to confirm this, indicating that once removed from the influence of BCC epithelium, BCC derived dermal fibroblasts tended towards senescence with continued passage. Quantitative Real, Time-PCR (qPCR) showed an increase in expression levels for markers of senescence. Immunocytochemistry of GUI showed expression in both normal dermal fibroblasts and BCC derived fibroblasts therefore qPCR was performed to detect the presence of the components of the Hedgehog signalling pathway. Treatment of the cells with an inhibitor of this pathway proved ineffective in reducing the expression of GUI. Therefore the presence of GUI in the BCC derived fibroblasts suggests that paracrine signalling may be important to the maintenance of the BCC stroma and hence BCC tumour growth. However the expression of GUI may be a result of a non-canonical pathway rather than the traditional Hedgehog Pathway. |
