| Objective:In this experiment,the photoaging model of human skin fibroblasts(HDFs)induced by UVA was established.CGF was applied to the HDFs damaged by UVA.Primary cell culture,immunofluorescence,flow cytometry,and molecular biology techniques were performed to detect the HDFs proliferation activity in vitro,the total amount of intracellular reactive oxygen species(ROS),the m RNA and protein expressions of P38mitogen-activated protein kinase(P38MAPK),c-jun amino-terminal kinase(c-jun),and matrix metalloproteinase-1(MMP-1).And then the effects of CGF on the HDFs damaged by UVA and the related mechanism of action were discussed.Methods:1. Primary culture and identification of human skin fibroblastsThe normal eyelid skin tissues come from the volunteers for double eyelidplasty in the department of plastic and aesthetic surgery,the second hospital of Hebei Medical University.HDFs were cultured by tissue explants adherent method.The third generation fibroblasts were identified by morphological observation and immunocytochemistry against mouse anti-human vimentin monoclonal antibodies and mouse cytokeratin(CK)monoclonal antibody.2. Selection of the irradiation doseThe HDFs were irradiated with UVA at doses of 5J/cm~2?10J/cm~2?20J/cm~2?30J/cm~2respectively.After irradiation,the HDFs were cultured in complete medium(DMEM containing 10%FBS)for 24 hours.The cells of control group and UVA group were observed under an inverted microscope and immediately after irradiation and 24 h after irradiation and MTT assays were performed to select the proper irradiation dose.3.The application concentration of CGFThe complete culture medium containing CGF(5%,10%,20%,30%)was applied to the HDFs radiated by UVA.The morphological changes of the cells were observed after 24 hours.The optical density of the cells cultured for24 hours,48 hours and 72 hours were detected by MTT assay.4.Experiment groupingThe cells were divided into the following three groups:Blank control group:HDFs were cultured in the complete medium for 24hours after pseudo-irradiation(the treatment process of cells was the same as the following two groups,but no UVA irradiation).UVA group:HDFs were cultured in complete medium for 24 hours after20J/cm~2UVA irradiation.UVA+CGF group:HDFs cells were cultured in complete medium containing 20%CGF for 24 hours after 20J/cm~2UVA irradiation.5.Detection of the intracellular ROS level of HDFsHDFs were labeled with 2,7-Dichlorodi-hydrofluorescein diacetate(DCFH-DA).The fluorescence intensity of HDFs was observed with inverted fluorescence microscope in the dark,and the content of ROS was detected by flow cytometry.6.Expression of 38MAPK,c-jun and MMP-1 in HDFs.The m RNA of P38MAPK,c-jun and MMP-1 in HDFs was extracted and detected by RT-PCR??C_T relative quantitative method,and the protein of p38MAPK,c-jun and MMP-1 in HDFs was extracted and detected by Western blot.7.Statistical analysisSpss21.0 statistical software was used for analysis.The data of each group was expressed in the form of mean±standard deviation(?x±s).The normal variance of the measurement data was analyzed by oneway ANOVA.The variance data were nonparametric test.The test level was a=0.05,P<0.05,and the difference was significant.Resurts:1.Primary culture and identification of human skin fibroblastsAfter 5-7 days of culture,the cells crawled out from the skin tissue,and the cells'bodies were large.The cell morphology is spindles or stars with multiple processes,the nucleus is regular oval,and the nucleolus is large and obvious.After 3-4 days of continuous culture,the cells grew radially around the tissue.Immunocytochemical staining showed that mouse anti-human vimentin monoclonal antibody was positive and mouse CK monoclonal antibody was negative.2.Morphological observation of HDFs in different treatmentsHDFs in the non irradiation group:cells were complete,closely connected and arranged radially.HDFs after UVA irradiation:After irradiation,the cells swell,rupture and suspend slightly swelling.24 hours after complete medium culture,the number of cell fragments and suspended cells increased and the cell arrangement was irregular.HDFs in CGF group:compared with UVA group,the shape of HDFs is more regular,the degree of atrophy is reduced,and the number of suspended cells is reduced.3. Select UVA radiation dose and establish light aging modelMTT results showed that:compared with the non irradiation group,the difference of 10J/cm~2,20J/cm~2,30J/cm~2 UVA irradiation group was statistically significant(P<0.05).The corresponding cell survival rate was91%,84%,64%,41%.We choose 20J/cm~2 as the UVA dose of photoaging model in this experiment4. Screening of CGF concentration.The results of MTT showed that:the OD value of HDFs in the non irradiated group,the UVA group and the CGF group increased after 24 hours,48 hours and 72 hours.Compared with 20J/cm~2 UVA group,there was significant difference in OD between the non irradiation group and CGF treatment group(P<0.05).In the CGF group,the effect of 20%CGF was the best.5. ROS expression observation and content detectionThe inverted fluorescence microscopy showed that the fluorescence intensity of cells in blank control group was the weakest,in the UVA group increased significantly,and the expression of ROS was high.The fluorescence intensity in the UVA+CGF group was lower than that in the UVA group,and CGF inhibited the expression of ROS.The results of flow cytometry showed that the average fluorescence intensity of the blank control group,UVA group and UVA+CGF group were3.963±0.775,7.800±0.300 and 5.673±0.579.The difference between three groups was statistically significant(P<0.05).6. Expression of p38MAPK,c-jun and MMP-1RT-PCR results showed that p38MAPK,c-jun and MMP-1 m RNA expression in UVA group increased by 6.346,6.246 and 3.070 times,respectively,compared with blank control group(P<0.05).P38MAPK,c-jun and MMP-1 m RNA in UVA+CGF group were 3.783,1.563 and 1.989 times higher than that in blank control group,respectively,and there was no significant difference between UVA+CGF group and blank control group(P>0.05).There was significant difference between UVA group and UVA+CGF group(P>0.05).Western blot showed that the protein expression of p38MAPK,c-jun and MMP-1 was the highest in UVA group,which was statistically significant compared with the blank control group and UVA+CGF group(P<0.05).There was no significant difference between UVA+CGF group and the blank control group(P>0.05).Conclusion:1.CGF could promote the proliferation of HDFs radiated by UVA.2.CGF can reduce the production of ROS in HDFs radiated by UVA,thereby reducing the oxidative damage of cells.3.CGF can inhibit skin UVA-induced photoaging by inhibiting p38MAPK/AP-1 signal pathway and reducing MMP-1 expression. |
PDF Full Text Request