Intense Pulsed Light In Effects Of TGF-β1 Induced On The Proliferation Of Fibroblasts And Intervention Of ERK Inhibitor

Objectives1. To observe the impact of the transforming growth factor-(31 in cultured human skin fibroblasts proliferation.2. To discuss the role of intense pulsed light in the transforming growth factor-β1 mediated fibroblast proliferation and the study of extracellular signal regulated kinase (ERK) signaling pathway.Methods1. Remove the foreskin of the normal adolescent by circumcision, isolation and culture in primary fibroblasts of normal human skin, using keratin and typeⅠ,Ⅲcollagen staining to identify the fibroblasts.2. The trail was divided into three parts:Part 1:TGF-β1 concentration treatment group, by using different concentration of TGF-β1 treatment of the fibroblasts, using tetrazolium salt (MTT) assay the proliferation of cells in each group;Part 2:TGF-β1 concentration group:lOng/ml,5ng/ml,1.0ng/ml and Ong/ml; Time Group:lOng/ml of TGF-β1 was give to stimulate the cell; Extract protein at different timing:0 min,15 min,30 min,60 min and 120min. Determine the changes of phosphorylation of extracellular signal regulated kinase (p-ERK) with western blotting;Part 3:Inhibitor intervention group:after the treatment ofFibroblasts with the control group, TGF-β11.0ng/ml,00.0ng/m;,IPL dose 72J/cm2 group,ERK inhibitor PD98059+TGF-β1 10.0ng/ml group.72J/cm2+TGF-β1 10.0ng/ml group,PD98059+72J/cm2 Group and PD98059+72J/cm2+ TGF-β1 10.0ng/ml group, using tetrazolium salt (MTT) assay the proliferation of cells in each group; determine the changes of phosphorylation of extracellular signal regulated kinase (p-ERK) with western blot; Results1. Cultured fibroblasts were negative by keratin, positive byⅠ,Ⅲcollagen staining, cells consist form of fibroblasts.2. Fibroblasts treated with different concentrations of tranforming growth factor-β1 effect, Compare with control group, the concentration of TGF-(31 in each group was treated for 24h and 48h respectively. There is no significant difference under the 0.01 ng/ml,0.1 ng/ml,1.0ng/ml,50.0ng/ml concentrations, (P> 0.05). and TGF-β1 concentration in 10.0ng/ml, the cell proliferation activity was significantly higher than the control group(P<0.05).3. Compare with the control group:TGF-β1 concentrations and time group had p-ERK protein expression differences. With increasing doses of TGF-β1 and the effect of time, the protein levels of ERK phosphorylation gradually increased. the TGF-β1 10.0ng/ml, the p-ERK had highest level of expression. At 15 min, the phosphorylation of ERK protein expression levels began to increase. At 60 min, the p-ERK expression levels reached a peak. At 120 min, the expression of p-ERK attenuated.4. In the inhibitor intervention group, to compare with the control group:IPL dose 72J/cm2 group and 72J/cm2+TGF-β110ng/ml and TGF-β110.0ng/ml group, the activity of fibroblast proliferation has increased (P<0.01), also the phosphorylated extracellular signal regulated kinase expression level has increased. As for PD98059+TFG-β1 10ng/ml group, PD98059+72J/cm2 group, and PD98059+72J/cm2 +TGF-β110ng/ml group, in comparison with the control group, had a decrease in both cell proliferation activity (P<0.05) and the expression levels of phosphorylated extracellular signal regulated kinase.there is no significant difference under the other groups.Conclusion1. A large of doses of TGF-(31 up-regulated in cultured fibroblasts of the ERK signaling protein activity. TGF-β1-induced fibroblast increased levels of p-ERK may be dependent on activation of ERK pathway. 2. Intense pulsed light (IPL) can promote the in vitro prolife-ration of fibroblasts. One of the mechanisms may be achi-eved through the ERK signaling pathway.3. TGF-β1 and intense pulsed light may be synergistic interact with each other in culture fibroblast proliferation.