| Objective:To establish a photodamage model of HaCaT cells by UVA irradiation of HaCaT cells and apply CGF to cells after photodamage,and to explore the effect of CGF on the photodamage of HaCaT cells induced by UVA.Method:1.HaCaT cell cultureHaCaT cells were cultured in a sterile incubator at 37°C,5%CO2.The experiment was carried out when the degree of cell fusion reached 80%or more.2.UVA irradiation dose screeningThe doses of UVA were selected from 10J/cm2,20J/cm2 and 30J/cm2.The absorbance at 570nm of each group was detected by MTT method and the cell viability under different irradiation doses was calculated.3.CGF concentration screening5%,10%,15%,20%CGF was selected as the concentration of CGF treatment,and the absorbance at 570 nm of each group was detected by MTT method.The obtained data were screened for CGF concentration by statistical analysis.4.Experimental groupingAccording to the selected UVA irradiation dose and CGF concentration,the cells were divided into three groups:Control group:HaCaT cells were not exposed to UVA irradiation and cultured in DMEM for 24 h.Irradiation group:HaCaT cells were irradiated with UVA irradiation at a dose of 30 J/cm2 and cultured in DMEM for 24 h.CGF treatment group:HaCaT cells were irradiated with UVA irradiation at a dose of 30 J/cm2 and cultured in 5%CGF+DMEM for 24 h.5.Cell morphology observationThe morphological changes of the cells in the three groups were observed under an inverted microscope and immediately after irradiation and 24 h after irradiation.6.Expression of reactive oxygen species?ROS?in cellsThree groups of cells were observed under the fluorescence inverted microscope at a wavelength of 488 nm for 24 h after irradiation.7.Intracellular superoxide dismutase?SOD?activity assayThree groups of cells were used to measure the absorbance values of each group on a microplate reader at a wavelength of 550 nm,and the activity of SOD at 24 h after irradiation was calculated according to the formula.8.Cell nuclear transcription factor Nrf2 expression observationThree groups of cells were selected for Nrf2 as the target protein for immunocytochemical staining to detect the expression and distribution of Nrf2protein.9.Statistical analysisThe data were analyzed by SPSS21.0 statistical software.The data of each group were expressed as percentage or mean±standard deviation??x±s?.The normal variance of the measurement data was analyzed by oneway ANOVA.The variance and the count data were nonparametric.test.The test level was a=0.05,P<0.05,and the difference was significant.Result:1.UVA irradiation dose screeningThe absorbance values of UVA irradiation doses of non·irradiated group,10J/cm2,20J/cm2,30J/cm2 were 0.274±0.035,0.241±0.009,0.238±0.013,0.199±0.016,respectively,and the corresponding cell survival rates were87.91%,86.86%and 72.53%.The OD value of the 30J/cm2UVA irradiation group was statistically different from that of the nonirradiated group?P<0.05?,and 30J/cm2 was selected as the subsequent experimental irradiation dose.2.CGF concentration screeningThe absorbance values of the unirradiated group,30J/cm2 group,5%CGF group,10%CGF group,15%CGF group and 20%CGF group were 0.223±0.014,0.157±0.012,0.253±0.026,0.222±0.017,0.221±0.011,0.199±0.017,r espectively.When the concentration of CGF was 5%?10%and 15%,the cell absorbance value was statistically different from that of 30J/cm2?P<0.05?.The cell absorbance value was the highest at 5%and 5%was selected as the CGF test concentration.3.Cell morphology observationThe cells in the control group were polygonal in shape and arranged closely.Immediately after irradiation,the cells shrunk and the cell gap increased.After 24h,the cells in the UVA group became smaller and narrower,and the intercellular space gradually increased.The cell morphology of the CGF treatment group was lower than that of the irradiated group,and the intercellular gap was decreased.4.Reactive oxygen species ROS expression observationThe fluorescence intensity of the control cells was weak.The fluorescence intensity in the irradiated group was significantly increased and the expression of ROS was increased.The fluorescence intensity of the cells in the CGF treatment group was lower than that in the irradiated group,and the fluorescence intensity of individual cells was high.5.Detection of superoxide dismutase SOD activity in cellsThe intracellular SOD activities of the control group,the irradiation group and the CGF treatment group were 32.350±1.116,24.457±2.443,29.846±0.588,respectively and there were statistical differences in each group?P<0.05?.6.Nuclear transcription factor Nrf2 expression observationNrf2 was mainly expressed in the cytoplasm in the control group.Part of the Nrf2 protein was transferred from the cytoplasm to the nucleus in the irradiated group,and Nrf2 was expressed in both cytoplasm and nucleus.Compared with the irradiated group,the expression of Nrf2 in the CGF treatment group was enhanced and the unclear accumulation was significantly increased.Conclusion:1.UVA irradiation can cause damage to HaCaT cells.2.CGF may increase the activity of SOD and eliminate excess ROS in the cell by promoting the transfer of Nrf2 from the cytoplasm to the nucleus of the cell,thereby increasing the activity of HaCaT cells and repairing damaged cells. |
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