Concentrated Growth Factors Can Inhibit Photodamage Induced By UVA On The Human Dermal Fibroblasts In Vitro

Objective:To establish skin cell photodamage model by skin fibroblasts repeatedly irradiated by the long ultraviolet wave,Concentrated Growth factor(CGF)was applied to human skin fibroblasts in skin cell photodamage model.We investigated the CGF’s capacity of scavenging reactive oxygen species,promoting proliferation and migration,and improving superoxide dismutase(SOD)in fibroblast cells irradiated by UVA.Methods:1.The culture and identification of human skin fibroblastsNormal human back skin tissue was obtained from patients in 25-30 old years,the 3rd Hospital,Hebei Medical University.This study was approved by the Ethics Committee of the Hospital of Stomatology,Hebei Medical University.The fibroblasts were cultured by using traditional method of primary culture in vitro.The third generation fibroblasts were identified by immunocytochemistry against mouse anti-human vimentin monoclonal antibodies and mouse cytokeratin(CK)monoclonal antibody,and collagen type III polyclonal antibodies.2.CGF Conditioned MediumVenous blood was obtained from the patients and was centrifuged in Vacuette tubes with green bottle cap.After centrifugation,the plastic straw draws CGF liquid.After freezing and filtration of the CGF liquid,the complete medium were added.Four CGF conditioned medium concentrations(10%FBS)were used:5%,10%,15%and 20%conditioned medium..3.UVA Treatment.Fibroblasts in the logarithmic phase were irradiated by a desktop apparatus for UVA photodamage group with a spectrum from 320 to 400 nm as the light source.Fibroblasts were treated by the photodamage group dose of18J/cm~2.Then the mean absorbance values of control and photodamage group were obtained by MTT assay after 24h,48h and 72h of culture in order to establish fibroblasts photodamage.4.Screening of CGF Conditioned MediumAfter photodamage group,fibroblasts were cultured in complete medium and CGF(10%fetal bovine serum)at an respective concentration of 5%,10%,15%and 20%.The mean absorbance values(x±s)of each group was obtained by MTT assay after 24h,48h and 72h at 490 nm wavelengths in order to determine the application concentration of CGF.5.Classification of the experimental group:Control group:fibroblasts were cultured in complete medium.Photodamage group:fibroblasts were irradiated with UVA at a dose of18J/cm~2 and cultured in complete medium.CGF treatment group:fibroblast was irradiated with UVA at a dose of18J/cm~2,and was cultured in 5%CGF.6.Detecting the number of ROS cellsThe cells in each group were marked by using DCFH-DA.The stained cells were imaged and analyzed randomly by using the Fluorescence microscopy in the dark and following the instruction of the ROS reagent kits.7.Detection of cellular SOD AssayAfter the cell supernatant of the three groups were collected,the activity of SOD was measured by hydroxylamine method.The enzymatic activity of superoxide dismutase was measured by the instruction of reagent kits(Nanjing Institute of Jiancheng Biological Engineering,China).All data are expressed in terms of SOD unit activity per milliliter.8.Scratch assayAfter cells/well reaching 90–100%confluence,a wound was generated in the confluent monolayer by scratching the monolayer with a 20-ul pipette tip.Fibroblasts migrating into scratched region were respectively photographed and measured on 24h,48h,72h,96h.9.Statistical analysesData were analyzed using One-way analysis of variance(ANOVA)and Student-Newman-Keuls’test.All data were processed with SPSS 21.0statistical software and were expressed as mean±standard deviation.P<0.05was considered as significant.Results:1.The culture and identification of human skin fibroblastsAfter 5-7 days of primary cell culture,cells emigrated from the tissue blocks,long spindle with 2-3 nucleolus,spherical or elliptic nuclei,uniform cytoplasm.After 9-13 days the cells began to converge with spiral radial growth.When the cells fusion rate reached 70–80%,fibroblast-like cells were passaged.The third generation of cells were used for immunohistochemical identification:The subcultured fibroblast-like cells were microscopically positive for collagen III and vimentin.CK in fibroblast-like cells reacted negatively by comparison.2.The establish of fibroblasts photodamageThere were significant difference in the value of OD between photodamage group and control group(P<0.05)after 24h and 48h.The results show that the radiation dose of 18J/cm~2 could cause the photodamage of fibroblasts.3.Screening of CGF Conditioned MediumThere were significant difference in the value of OD between CGF treatment group and other groups(P<0.05)after 24h and 48h.Because the5%CGF group has the highest OD value,5%CGF was selected for the following experiments.4.Cell viability of CGF on UVA-treated fibroblastsAfter 24h of culture,the cell viability of two groups were 0.4482±0.0603(group photodamage group),0.601±0.035(group CGF treatment),respectively.There were significant difference between two groups(P<0.05).After 48h of culture,the cell viability of two groups were 0.6748±0.0918(group photodamage group),0.7457±0.2513(group CGF treatment),respecti-vely.There were significant difference between two groups(P<0.05).5.Detecting the number of ROS cellsThe number of ROS cells in each group:The number of ROS cells in the photodamage group,CGF treatment group and control group were65.5556±3.3582,22.7778±2.4381 and 3.3333±1.8028,respectively.There were significant difference among three groups(P<0.05).6.Effect of CGF on cellular SODThe activities of SOD(U/ml)in each group:The activities of SOD(U/ml)in the photodamage group,CGF treatment group and control group were22.4725±1.2656,31.8462±2.3332 and 37.1833±2.5315,respectively.There were significant difference among three groups(P<0.05).7.Scratch assayThe migration rates of the photodamage group group,CGF treatment group and control group were 2.35±0.84%,74.9±3.17%and 62.07±4.23%,respectively,after culturing for 24h.There were significant difference among three groups(P<0.05).Conclusions:1.UVA photodamage group can cause the photodamage of the fibroblasts.2.CGF can reduce the level of ROS in cells and improve the activity of SOD after UVA photodamage group,and can inhibit photodamage induced by UVA in human skin fibroblasts.3.CGF can improve the activity of fibroblasts after UVA photodamage group and promote proliferation and migration.