| Objective:Skin photoaging is a phenomenon where long-term exposure to excessive ultraviolet radiation leads to the formation of spots and wrinkles on the skin.Currently,the main method for preventing and treating skin photoaging is the use of sunscreen products,but the quality of these products on the market varies greatly.Therefore,this study aims to explore the protective effect of melanin granules derived from melanocytes against UVB-induced skin photoaging and to find a more effective and safe method for preventing skin photoaging.Methods:1.In the in vitro test,CCK8 and Hoechst3342 staining was used to draw the standard curve.After irradiating Ha Ca T cells with different doses of UVB,the morphological changes of Ha Ca T cells were observed under microscope,and the cell viability of each dose group was measured by CCK8 method.2.By HE staining,Masson-Fontana staining and immunofluorescence staining of normal human foreskin tissue.Primary melanocytes and keratinocytes were isolated from normal human foreskin tissues and microscopic morphological observation to identify the expression of melanocytes and keratinocytes-specific genes by q RT-PCR.3.By observing the morphological characteristics of human epidermal melanocytes(HEM)and melanoma cells(A375)through microscopy;by comparing the melanin granules of the two types of cells and their cells using Masson-Fontana staining,L-DOPA staining,CCK8,and q RT-PCR.4.The extracted melanin granules were added to Ha Ca T cells and keratinocytes(KC)medium,and the distribution of melanin granules in cells was observed by Masson-Fontana staining after 24 hours of culture.The protective ability of melanin granules against UVB-induced photoaging of keratinocytes(KCs)was detected by CCK8 and q RT-PCR.Results:1.Irradiating Ha Ca T cells with different doses of UVB(0,60,180,300,420,540 m J/cm~2),when the UVB dose increases,the cell morphology changes or even falls off and dies,and the cell survival rate gradually decreases.Finally,180 m J/cm~2 UVB irradiation cells were established.2.Normal human melanocytes and keratinocytes were successfully isolated,melanocytes can express TYR,TYRP1,MITF,keratinocytes can express KRT1,KRT10,KRT5,KRT14,KRT15.3.Human epidermal melanocytes(HEM)Masson-Fontana staining and L-DOPA staining were positive.Compared with melanoma cells(A375),the gene expression of TYR and TYRP1in HEM was significantly increased.After centrifuging the cell supernatant,select 20μm filtration membranes for filtration to obtain melanin granules.HEM-derived melanin content is significantly higher than A375.4.Both Ha Ca T cells and keratinocytes(KC)can engulf melanin granules,and melanin granules appear obvious cap shape around the nucleus in keratinocytes(KC).Compared with the UVB group,the melanin granule group significantly improved the viability of cells.Compared with the unirradiated cells,the expression of KRT5,KRT18 and KLF6 genes in the UVB group decreased significantly.Compared with the UVB group,the expression of KRT5,KRT15,KRT18 and KLF6 genes in the melanin granules group was significantly increased.Conclusion:Melanin granules can reverse the decrease in keratinocyte viability induced by UVB,and upregulate the expression of dryness-associated genes(KRT5,KRT15)and aging-associated genes(KRT18,KLF6)in the keratinocytes,thus providing protection against UVB-induced cellular photoaging.This study creatively explored the protective effect of melanin granules on skin photoaging using the natural endogenous substance and promoted the application of melanin granules in skin beauty and sun protection,providing new ideas for preventing skin photoaging. |
