Development of a cell based chronic wound bioassay

Impaired wound healing affects 3% of the population over 60 and costs over £1 billion annually in the UK. Chronic wounds are characterised by prolonged inflammation, impaired re-epithelialisation and defective extracellular matrix (ECM) remodelling. Fibroblasts play an important role in the closure of skin wounds, they replace and remodel the lost tissue and also influence both re-epithelialisation and angiogenesis. Numerous in vivo chronic wound animal models have been reported however, they fail to accurately model human chronic wounds. This investigation aims to develop an in vitro chronic wound bioassay. Chronic wound fibroblast (CWF) and patient matched normal fibroblast (NF) cell strains (n=3) were retrovirally infected with a hTERT/puromycin construct. Population doubling levels (PDLs) were determined over an extended time in culture. RNA at defined time points was extracted and analysed using Asymetrix U133A microarrays. A cohort of gene expression changes were confirmed by QRT-PCR and the promoter regions of these genes used to construct fluorescent reporter human NF and CWF cell lines. Population doubling levels indicated that, compared to primary (non-immortalised) CWF and NF, the hTERT infected fibroblasts had escaped replicative senescence and formed an immortalised cell line. Affymetrix microarray analysis of the CWF-hTERT with the NF-hTERT identified 247 genes that were significantly up- or down-regulated in CWF (validated by QRT-PCR). Upstream promoter regions of 13 genes of interest and a housekeeping gene have been identified by database searches/sequence analysis, amplified and cloned into the promoterless reporter vector pZsGreen1-DR. Reporter constructs have been transiently transfected into human NF and CWF cell lines and are currently being tested for efficacy. CWF and NF cell lines have been created which demonstrated distinct gene expression profiles. The disease marker genes will form the basis of a low cost, highly reproducible fluorescent reporter cell-based bioassay which will be utilised in the discovery of novel therapeutics which have the potential to alter the chronic wound healing and, in turn, reduce unnecessary animal experimentation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo