The Effect Of Mitomycin On Cultured Human Normal Skin Fibroblast And HaCat Cell

Objective:To investigate the effects of mitomycin on cultured human normal skin fibroblast and HaCat cell,including cellular morphology,cell proliferation,growth characteristics and apoptosis,in order to find out the clinically relevant concentrations of mitomycin.With the concentration,we try to detect the expression changes of intracellular caspase-3,capase-8,TGF-β1 on protein level and TGF-β1,bFGF, procollagenⅠand procollagenⅢon mRNA level separately,which can give theoretical supports to the clinical application of mitomycin.Methods:Cultured human normal skin fibroblast and HaCat cell in vitro,mitomycin was applied to the cells for 5 minutes with concertrations of 0.04mg/ml or 0.4mg/ml,and cultural media without serum was used as control,then the cellular morphology change,growth characteristics,cell proliferation were observed by inverted microscope,AO staining,cell count and MTT test at different intervals.The apoptosis rate of fibroblast was detected with AO/PI staining.For fibroblast,western blot was used to detect caspase-3 expression.Immunofluorescense was used to detect caspase-8 activation and intracellular TGF-β1 expression.RT-PCR was used to detect the mRNA expression of TGF-β1,bFGF,procollagenⅠand procollagenⅢ.IPP was used to analyze picture and SPSS 15.0 was used to make statistical analysis. Result:The cultured normal human skin fibroblast and HaCat cell grew with a exponential phase.Mitomycin could inhibit the proliferation of both fibroblast and HaCat cell dose-dependently,and it could make the cells grow bigger and irregular.5 days after the 5min exposure of mitomycin at 0.04mg/ml,fibroblast got a tiny proliferation,the inhibition effect of mitomycin at 0.04mg/ml was weaker than 0.4mg/ml(p＜0.05). Meanwhile,application of mitomycin at either 0.04mg/ml or 0.4mg/ml for 5min induced fibroblasts apoptosis but not necrosis.the higher concentration of mytomycin is,the higher apoptosis rate of fibroblast is (p＜0.05).After 0.4mg/ml mitomycin's application,the quantity of intracellular pre-caspase-3 rose at first,and then fell down. Immunofluorescense test showed that:the immunofluorescense intensity of active caspase-8 increased gradually after mitomycin's application,and there is no significant change in TGF-β1's immunofluorescense intensity. But the mRNA level of TGF-β1 decreased obviously after mitomycin's application,as well as procollagenⅠand procollagenⅢ.In contrary, bFGF increased on mRNA level.Conclusion:Mitomycin could inhibit fibroblast proliferation and increase its apoptosis through activating caspase pathway.Mitomycin could regulate the expression of TGF-β1 and bFGF,and decrease procollagenⅠandⅢexpression on mRNA level,which might reduce extracellular matrix synthesis and lessen scar formation.But mitomycin had even stronger inhibition effect on HaCat cell,so we sould pay more attention to protect epithelial cell to avoid mitomycin's side effect.