The Significance Of SPRY1 Gene On Senescence Of Keratinocytes

BackgroundAging is the continuous accumulation of degenerative changes in the body and is a complex physiological process.Skin aging is the result of a combination of endogenous factors?natural physiological aging?and exogenous factors?environmental effects?,which is manifested as slowed epidermal renewal,weakened barrier function,decreased keratinocyte activity,and decreased self-repair ability after epidermal injury.During the aging process,due to the action of free radicals and peroxidation,the surface of collagen forms AGE?terminal saccharification product?and crosslinks excessively,which makes the skin hemidesmosome structure lose its elasticity,the epidermal dermal connection becomes flat,and the skin becomes slack and loses elasticity that influence the appearance of the skin.Nowadays,skin aesthetics has become an important indicator of external aesthetics and has attracted wide attention.The aging of skin appearance is reflected in cell senescence.Cell senescence means that the normal cells have only a certain life cycle and cannot proliferate indefinitely.In the study of aging,people pay more attention to the aging of skin fibroblasts.In recent years,it has been gradually recognized that the aging of keratinocytes is an important manifestation of skin aging,because it plays a very important regulatory role in skin hydration,moisturizing,barrier and the formation of skin diseases.In the theory of skin aging,oxidative stress aging is one of important theories.Hydrogen peroxide?H₂O₂?can cause sustained oxidative stress,lead to cell damage and apoptosis.Therefore,H₂O₂ is often used to establish a cellular oxidative aging model,which is a safe and efficient method for inducing cell senescence in vitro.The SPRY1 protein is a cysteine-rich protein found in the study of Drosophila gene spectrum,and is an important biomolecule that antagonizes signaling pathways such as receptor tyrosine kinase?RTK?.The SPRY1 protein is regulated by some growth factors,such as fibroblast growth factor?FGF?,epidermal growth factor?EGF?and so on.At the same time,SPRY1 also plays a negative feedback regulation role on the above cytokines.As a negative regulator of various growth factor signaling pathways,the SPRY1 protein plays an important role in the proliferation,invasion,migration and apoptosis of various types of tumor cells.At the same time,SPRY1 is involved in the proliferation and aging of muscle stem cells.There has been a recent study on the function of SPRY1 in human keratinocytes and epidermis.There is no direct study on the expression of spry1 in skin keratinocytes and aging HaCaT cells at different age groups.Therefore,the study aims to visually explore the aging correlation between SPRY1 and keratinocytes,in order to find new ideas for skin aging and rejuvenation research.PART?THE EXPRESSION OF SPRY1 IN NORMAL HUMAN EPIDERMAL KERATINOCYTES OF DIFFERENT AGESObjectiveTo detect the expression of SPRY1 in normal human epidermal keratinocytes?NHEK?of different groups which include the minor group?<18 years old?,the youth group?18-44 years old?,the middle-aged group?45-59 years old?,and the elderly group??60 years old?.Methods1)The expression of SPRY1 mRNA in NHEK of different groups were detected by Real-time PCR.2)The cellular location and expression of SPRY1 in NHEK of different groups were detected by immunohistoch-emistry.3)The expression of SPRY1 in NHEK of different groups were detected by Western blot.Results1)The relative expression of SPRY1 mRNA in the tissues of minor,youth,middle-aged and elderly groups were 1.07±0.39,2.23±0.54,3.22±0.73,4.45±1.40?F=84.402,P=0.000?,respectively.The expression of the minor group was the lowest,and the expression of the elderly group was the highest,suggesting that the expression of SPRY1 mRNA in human skin keratinocytes increased gradually with the growth of age.2)The expression of SPRY1 in NHEK is mainly concentrated in the spinous layer and granular layer,showing cytoplasmic distribution,and no obvious expression in other epidermal layers and dermis.The percentage of positive cells and staining intensity increased significantly from the minor group to the elderly group.3)The expression of SPRY1 protein was observed in the minor group,the young group,the middle-aged group and the elderly group.The relative expression of SPRY1 protein in each group was 0.35±0.10,0.45±0.10,0.78±0.09,1.04±0.21?F=163.228,P=0.000?.It is suggested that the expression of SPRY1 protein increases with age.Conclusions1)The expression of SPRY1 is mainly concentrated in the spinous layer and the granular layer in NHEK.2)SPRY1 gene is closely related to the natural aging process of NHEK.PART? THE EXPRESSION OF SPRY1 IN NORMAL AND AGING GROUPS OF HACAT CELLS Objective To investigate the relationship between SPRY1 gene and HaCaT cell senescence by constructing and verifying the aging model of HaCaT cells and then detecting the expression of SPRY1 in different groups of HaCaT cells.Methods1)The construction of normal and aging HaCaT cells: normal group?without special treatment?,aging group?the cells cultured with200?mol/L H₂O₂ for 1 hour?.2)To verify the successful construction of HaCaT cell aging group,we detected the cell proliferation rate,T-SOD activity,and the expression of P21,P27,CDK2,Cyclin E in the normal group and aging group of HaCaT cells.3)The expression of SPRY1 mRNA and protein in normal and aging groups of HaCaT cells was detected by Real-time PCR and Western blot,respectively.Results1)The proliferation of HaCaT cells in the aging group was lower than that in the normal group by CCK-8 assay.The T-SOD activity of the normal group and aging group were 17.27±0.3,25.38±2.14?t=-12.983,P=0.000?,respectively,and the viability of the aging group was increased.The Western blot results of cell cyclin-related proteins P21,P27,CDK2 and Cyclin E showed that the expressions of P21?21kD?and P27?27kD?were up-regulated,while the expressions of CDK2?34kD?and Cyclin E?47kD?were decreased after HaCaT cells were aged.2)The relative expression of SPRY1 mRNA in the normal group and age group were 1.03±0.27,4.11±1.13?t=-7.917,P=0.000?,respectively.The relative expression of SPRY1 protein in the normal group and age group were 0.49±0.05,0.77±0.07?t=-5.527,P=0.005?,respectively.The expression levels of the aging group were higher than those of the normal group.Conclusions1)The HaCaT cells were cultured at 200?mol/L H₂O₂ for 1 hour to induce aging,and the HaCaT aging model was constructed successfully.2)The expression of SPRY1 mRNA and protein in HaCaT cells of aging group was up-regulated compared with the normal group,suggesting that SPRY1 may be involved in the regulation of senescence of HaCaT cells.