In Vitro Induce HaCaT Cell Apoptosis By SRB Assay And The Effect Of Ginsenoside On Apoptosis

Recent studies indicated that keratinocyte (KC) apoptosis is the initiating event in the development of the epidermal pathology seen in eczematous dermatitis. Apoptosis of KC leads to the loss of intercellular cohesion (acantholysis). Fluid influx from the dermis and intercellular edema contributes to spongiosis, which is defined as widening of the intercellular space and a spongelike appearance of the epidermis. It can injure the barrier function of skin and exacerbate eczematous dermatitis. Thus, in the process of chronic eczematous dermatitis, the apoptosis of keratinocyte is not only a turning point, but also a new target of drug screening. Flow cytometry is a sensitive and veracious method to analyze cell apoptosis, but it does not fit high-throughput drug screen because it need a lot of cells and reagents. Sulforhodamine B (SRB) and methyltetrazolium (MTT) assays are rapid, reproducible and sensitive assays to assess cells growth inhibition in 96-well plate. We can use these assays to preliminary screen anti-apoptotic drugs if we can discriminate the growth inhibition caused by necrosis and/or apoptosis. I n this research apoptosis of keratinocyte induced by interferon-γ(IFN-γ) and anti-Fas monoclonal antibody (antiFas Ab), mimicking down stream in the cascade of the pathogenesis of eczematous dermatitis, was used as an in vitro experimental model to screen drugs which can inhibit cell apoptosis. Then we compared the MTT and SRB assays and found that SRB assay was a more sensitive and stable assay to assess KC viability in vitro. To confirm that the inhibition of cell growth detected by SRB assay was not due to necrosis but apoptosis, cell apoptosis was examined by flow cytometry.Methods: The cultured keratinocyte HaCaT was exposed to...