| Research backgroundMesenchymal stem cells(MSCs)have strong ability of self-replication,renewal and multi-directional differentiation,and are an important treatment for self-repair after injury.Mesenchymal stem cells were first found in bone marrow and later in umbilical cord,cord blood,placenta,fat,periodontal,muscle and other tissues.At present,adipose-derived stem cells(ADSCs)and bone marrow mesenchymal stem cells(BMSCs)are the most studied and widely used in clinical practice,especially ADSCs,because of their wide range of sources and easy extraction of adults.Therefore,ADSCs have been widely used in the field of plastic and aesthetic,it works in the following ways:fill facial soft tissue depression,skin anti-aging,improve facial wrinkles,breast and hip augmentation,repair of refractory wounds.However,ADSCs also have some shortcomings and limitations:adult stem cells have limited activity,aging is obvious after multiple generations of culture,and the older age of some wound patients affects the activity of ADSCs;it has strong immunogenicity and can only be applied autonomously at present;the ability of multi-lineage differentiation is not as strong as that of embryonic or fetal stem cells.Compared with adult mesenchymal stem cells,fetal mesenchymal stem cells have the following advantages:higher self-proliferation ability,slower aging rate after multiple generations,stronger multi-directional differentiation ability,and lower immunogenicity.Fetal dermal mesenchymal stem cells(FDMSCs)were extracted from fetal dermal tissue in the second trimester of accidental abortion have potential advantages in the fields of skin regeneration and anti-aging.As for the mechanism of mesenchymal stem cells,many research results have shown that stem cells play a role mainly through paracrine function.Stem cells can release a series of bioactive substances such as cytokines,inflammatory mediators,growth factors,exosomes and so on,and play a variety of biological effects through the action of these mediators.The conditioned medium(CM)of stem cells contains the above media,which is an important means to study the mechanism of stem cells.In recent years,the anti-aging research of organisms or cells has attracted extensive attention of scholars.Among them,the anti-aging research of skin has attracted much attention in the field of plastic and cosmetic surgery.D-galactose is widely used in the establishment of cell senescence models and animal senescence models.The mechanism of accelerating senescence is very complex and has not been fully studied.However,the research on anti-aging effect of FDMSCs is still blank,so this research is carried out.Research purposeADFs senescence model was established by using D-galactose,and skin senescence model of mouse was established.To study the effect of FDMSCs-CM on the function of ADFs and its anti-aging effects in vivo and in vitro experiments,and to elucidate some of the mechanisms of FDMSCs-CM anti-aging effects.Research methods1.Extracting FDMSCs.FDMSCs were extracted from fetal dermis,and differentiation of adipogenic,osteogenic and chondrogenic lines was induced,and cell surface markers were detected.2.ADFs were extracted.ADFs were extracted from adult prepuce dermis and detected by Vimentin immunofluorescence.3.FDMSCs-CM extraction.4.To study the effects of different concentrations of D-galactose on ADFs and establish the aging model of ADFs.5.To study ADFs when FDMSCs-CM and D-galactose act together and observe the effect of FDMSCs-CM on ADFs proliferation,and study whether ADFs have anti-apoptosis and anti-aging effects in vitro.6.To study the effect of D-galactose on the content of reactive oxygen speciesin ADFs and the effect of FDMSCs-CM intervention7.To study the effect of 40g/L D-galactose on the expression of p16,p21 and p53 genes in ADFs and the effect of FDMSCs-CM intervention.8.Animal experiments.To establish a model of skin aging on the back of mice by subcutaneous injection of D-galactose..9.The survival time of FDMSCs in mouse back was studied by lentivirus transfection with luciferase gene.10.FDMSCs were injected subcutaneously to interfere with aging group Ⅱ mice to observe the anti-aging effect of stem cells.11.After injection of FDMSCs for 2 weeks,the mice were anesthetized and shaved for photos.Then the back skin was taken and stained with HE and MASSON to observe the skin tissue.Another part of the skin was made into homogenate to detect the content of components related to skin aging:SOD,MDA,HYP and AGEs.Research result1.FDMSCs were successfully extracted and identified.2.ADFs were successfully extracted and identified.3.FDMSCs-CM were obtained by extraction.4.D-galactose inhibited the proliferation of ADFs,and the inhibition increased with increasing concentration.5.High concentration of D-galactose can obviously induce apoptosis of ADFs.6.FDMSCs-CM can resist the apoptosis of ADFs induced by high concentration of D-galactose to some extent.7.FDMSCs-CM promotes the proliferation of ADFs and resists the inhibitory effect of low and medium concentrations of D-galactose on the proliferation of ADFs.8.40g/L D-galactose increased senescence associated β-galactosidase staining in ADFs,and inhibits ADFs proliferation significantly,thereby establishing ADFs senescence model.While FDMSCs-CM partially improved ADFs senescence.9.D-galactose induced a concentration-dependent increase in ROS in ADFs,FDMSCs-CM reduced the increase in ROS to a certain extent.10.The increase of p16,p21 and p53 gene expression was induced by 40g/L D-galactose,which was partially reversed by FDMSCs-CM.1 1.Prolonged subcutaneous injection of D-galactose induced skin aging in mice.12.FDMSCs survived subcutaneously in KM mice for approximately 10-11 days.13.Subcutaneous injection of FDMSCs can reduce skin wrinkles,increase dermal thickness,enhance SOD activity in skin of aging mice,reduce MDA and AGEs content,and increase HYP content in skin of aging mice.Research conclusion1.40g/L D-galactose can inhibit ADFs proliferation and induce ADFs senescence.2.FDMSCs-CM can promote ADFs proliferation and reverse ADFs senescence induced by 40g/L D-galactose.3.80g/L D-galactose can induce apoptosis of ADFs,and FDMSCs-CM can resist this apoptosis in certain extent.4.D-galactose may induce senescence and apoptosis by increasing the oxidative pathway of ROS level in ADFs,thus increasing the expression of p16,p21 and p53 genes.The above situation can be partially reversed by FDMSCs-CM,indicating that FDMSCs play an anti-aging effect at the cellular level mainly through antioxidant pathway.5.Subcutaneous injection of D-galactose can induce skin aging in mice,while subcutaneous injection of FDMSCs has a certain anti-aging effect,which is mainly through paracrine mechanism.Research significanceFDMSCs are relatively novel mesenchymal stem cells,and there is no literature report on anti-aging effect of FDMSCs in the world.In this experiment,the aging model of ADFs was established by using D-galactose at a certain concentration and induction time,and it was confirmed that FDMSCs-CM has anti-aging effect in vitro.Then,a mouse skin aging model was established by using D-galactose,and it was confirmed that FDMSCs can survive for more than 10 days in mice,and works normally display the anti-aging function,which reduced wrinkles,increased dermal thickness,increased collagen content,and decreased the content of various aging markers in the aging group.Therefore,this experiment proves that FDMSCs have certain anti-aging effects in both vivo and vitro.The anti-aging effects in vitro are mainly achieved through antioxidant pathways,while the anti-aging effects in vivo are mainly through paracrine mechanisms.The results of this experiment indicate that FDMSCs may be of great value and significance in establishing cell bank and promoting various clinical applications in the future. |
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