Research On Exosomes Of Mesenchymal Stem Cells In The Treatment Of Skin Aging

Photoaging not only results in external signs such as dryness,sagging,and wrinkles,but can also lead to various skin diseases including chronic actinic dermatitis,basal cell carcinoma,and squamous cell carcinoma.Unfortunately,current clinical treatments such as Chemical Peel,hya-luronic acid filling,radiofrequency and laser irradiation have limited effectiveness and are often accompanied by pain and bruising.Hence,it is crucial to explore and develop safer and more efficient treatments.Exosomes derived from mesenchymal stem cells（MSCs-Exos）have become a research hotspot for the treatment of skin ageing and tissue repair because of their excellent anti-inflam-matory and antioxidant functions.Under conventional culture conditions,the yield of MSCs-Exos is limited and varies significantly in terms of active substances and functions among differ-ent sources,which hinders its application and widespread use.However,interventions such as drugs,cytokines,and hypoxia can promote the secretion of exosomes and enrich them with spe-cific components,thereby improving the efficacy of disease treatment.To enhance the effectiveness of MSCs-Exos in treating skin photoaging,we prepared mes-enchymal stem cells derived from umbilical cord and adipose tissue（UCMSCs and ADSCs）.We then compared the expression differences of anti-aging related micro RNA,such as mi R-93,mi R-106,and mi R-146 in their exosomes,and selected candidate exosomes with greater anti-aging potential for further research.Melatonin was used in combination with hypoxic culture mode stimulate candidate exosome-derived parent cells to optimize the yield and efficacy of exosomes in treating skin aging,and make them have the potential to be developed into a Category I Bio-logical product for treating skin aging and related diseases.Primary UCMSCs were extracted from the umbilical cord by tissue culture method,while ADSCs were extracted from adipose tissue enzyme digestion method respectively.These cells were then passaged and cultured in a ten-layer cell factory to successfully construct a P4 gener-ation UCMSCs and ADSCs seed cell bank.The results of immunophenotypic and multi-direc-tional differentiation indicate that the P4 generation UCMSCs and ADSCs are MSCs.The ex-tracted exosomes were characterized by Transmission Electron Microscope（TEM）,Nanoparticle Tracking Analysis（NTA）and Western Blot,respectively.The results indicate that the UCMSCs-Exos and ADSCs-Exos extracted through ultrafiltration combined with differential centrifugation were exosomes.The q PCR experiment was conducted to examine the difference in the levels of anti-aging-related micro RNAs,specifically mi R-93,mi R-106,and mi R-146,present in both UCMSCs-Exos and ADSCs-Exos.The results suggested that ADSCs-Exos possess greater anti-aging potential,making them the preferred choice for further investigation.The effects of combining melatonin（MT）stimulation with hypoxia culture（1%O₂）on the biological properties of ADSCs and secretion of ADSCs-Exos were evaluated using various tech-niques,including CCK8,NTA,immunophenotype identification,and multi-directional differen-tiation.The results showed that MT stimulation promoted the secretion of ADSCs-Exos.Specif-ically,in which 20μmol/L melatonin stimulation for 48 h resulted in a 3.4-fold increase in the yield of ADSCs-Exos.Importantly,this stimulation did not affect the immunophenotype,apop-tosis,or multi-directional induction potential of ADSCs.Moreover,the compound stimulation of MT combined with hypoxia can further improve the yield of ADSCs-Exos,and promote the se-cretion of more vascular endothelial growth factor（VEGF）and platelet-derived growth factor（PDGF-AA）by ADSCs,which are related to tissue repair.The effects of melatonin stimulation combined with hypoxic culture-generated ADSCs-Exos（H-MT-Exos）on UVB-induced cellular senescence were investigated byβ-galactosidase（SA-β-gal）staining analysis,cell cycle detection,cell scratch,ELISA and q PCR.The results of SA-β-gal staining analysis,cell cycle detection and scratch experiments showed that UVB irra-diation dose of 300 m J/cm² successfully induced cell senescence and lead to cell cycle arrest,while pretreatment with 20μg/m L H-MT-Exos could reduce cell senescence caused by UVB and promote cell migration.q PCR and ELISA results showed that pretreatment with H-MT-Exos sig-nificantly reduced the production of ROS after UVB irradiation,inhibited the m RNA expression of cell cycle regulators p16^INK4a,p21^Cip1,p53,matrix metalloproteinases MMP-1 and MMP-3,and promoted collagen type I synthesis and secretion.In brief,UCMSCs and ADSCs isolated by tissue culture and enzyme digestion have prop-erties of MSCs,and UCMSCs-Exos and ADSCs-Exos with exosome characteristics can be ex-tracted by ultrafiltration combined with differential centrifugation,and ADSCs-Exos have more anti-aging potential.The combined stimulation of melatonin with hypoxic culture can optimize the yield of exosomes and their efficacy in treating skin photoaging,providing a theoretical and practical basis for the development of MSCs-Exos into a Category 1 Biological Products for the treatment of skin aging and the prevention of skin aging-related diseases such as basal cell carci-noma.