Establishing The Apoptotic Model Of HaCaT Cell Induced By UV And A Primary Study On Protection Of HaCaT Cell Against UV

Objective:TO establish UVA Plus UVB-induced apoptotic model of HaCaT cells and investigate the mechanism of polypeptide from EGCG protecting HaCaT cells from apoptosis induced by micking the action of environmental UV irradiation on human skin.Materials and methods:HaCaT cells were cultured in DMEM medium with 10%fetal bovine serum and cells were plated in 3.5cm dishs and 96-well plate with an equal number of cells(104-106 cells). Ultraviolet irradiation:An apoptotic model of UV irradiation-induced HaCaT cells was established by orthogonal design. Tested medicines were added into the medium before and after UV illradimion. Cell viability assay:The dose-and time-dependent photodamage was observed by light microscopy; methy thiazoly tetrazolium (MTT)assay was used to record cell proliferation and cellular activity. The apoptosis of HaCaT cells:The apoptosis rates were detected by TUNEL assay or flow cytometry, Also confirmed if the results of orthogonal designs is to establish the best apoptosis model. agarose gel electrophoresis and Hochest33258 staining show HaCaT cells from apoptosis Induced by UVA plus UVB and Intervention Role of EGCG from two pathways of morphology and biochemical characteristics. Statistical analysis:The experimental data were analyzed with SPSS. Comparisons between groups were tested by One-Way ANOVA analysis and LSD test.P values less than 0.05 were considered to be statistically significant.Results:1,At the early time of cell culture of HaCaT,they form round or oval adherent cells, and visible on the small spider shape or roots cells and eventually could be connected to a typical "piece stepping stones" shape.2,The result of orthogonal designs, MTT, Flow Cytometry test the apoptosis of cells, Agarose gel electrophoresis and Hoechst33258 show from the different aspect that the HaCaT cell apoptosis after 6J/cm2UVA+15mJ/cm2UVB irradiation was maximal and obviously Larger than the space group,The result showed that UVA+UVB obviously inhibited the cell activity of proliferation.3,The EGCG dose of 12.5-100μg/mL don't affected the cell activity, and was no damage and toxicity.The activity of HaCaT cell dealt with the different dose EGCG was more active than the Simply poured group.All this show that EGCG can protected HaCaT cell form UV radiation. UV radiation can reduced the cell activity, which was dose-correlation. The damage that UV irradiated HaCaT cell was reduced by EGCG. The concentration of 50μg/mL was the best choice.Conclusions:This study shows that UVA Plus UVB may induced apoptotic model of HaCaT cells,and that EGCG may reduce the rates of apoptosis.