CYTOKINE GENE EXPRESSION IN THE MATTEK EPIDERM CULTURES.
Human skin equivalent cultures are currently being investigated as in vitro models to predict the contact sensitization potential of chemicals. The objective of the research presented here is to evaluate the constitutive expression of a panel of immunomodulatory cytokine genes and to determine the modulation of these genes following exposure to phorbol myristic acetate (PMA) in one such culture system, the MatTek EpiDerm™ model. EpiDerm cultures were topically treated with PMA (50 nM), assay medium, or no treatment for 2 or 4 hours, followed by total RNA isolation using Qiagen’s RNeasy kit. Cytokine gene expression was evaluated by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and liquid hybridization, with normalization to the housekeeping gene, G3PDH. Message for IL-1α, IL-β, IL-6, IL-8, and TNF-α was constitutively expressed at various levels in the MatTek EpiDerm model. IL-1α and IL-8 have the highest steady state levels of message, while IL-6 has the lowest levels. Gene expression was not detected for GM-CSF and IL-10. Treatment with PMA produced an increase in the steady state levels of message for several of the cytokine genes, while the housekeeping gene, G3PDH, was unaffected. No significant difference was seen between the untreated and media-treated groups. These studies demonstrate the EpiDerm model constitutively expresses message for a number of cytokines and message levels can be modulated by the addition of phorbol esters. Future studies with various sensitizers and irritants will determine if this is a viable model to identify sensitizers and differentiate them from irritants.
Chemicals, Contact sensitization, Cytokines, EpiDerm, G3PDH, housekeeping gene, Gene expression, Housekeeping gene, IL-1a, IL-1b, IL-6, IL-8, Immunomodulatory cytokine genes, Messenger RNA (mRNA), Pre-validation, Prevalidation, RNA isolation, TNF-a, Validation
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