| Objecive:Based on previous findings that Bone Marrow Mesenchymal Stem Cells（BMSCs）could reverse multi-organ aging in rhesus monkeys,we further studied the reversing effects of BMSCs on skin tissue structure and function in aged rhesus monkeys.By analyzing the skin proteomics data of rhesus monkey,candidate molecules of BMSCs to reverse skin aging were obtained.Through in vitro co-culture experiment of h UCMSCs and senile human skin fibroblasts,the effect of h UCMSCs on reversing cell senescence was clarified,and HAT1,a key molecule regulating senescence,was verified and screened.The key role of HAT1 in skin aging was verified by knockdown and overexpression of HAT1 in HSFs.Methods:1.Animal grouping:Rhesus monkeys of different ages were divided into juvenile group（n=5）,young group（n=5）,middle-aged group（n=3）,elderly group（n=4）and elderly-treatment group（n=6）.2.Evaluation method of reversing skin aging with BMSCs:Rhesus monkeys in the aged treatment group were injected with BMSCs intravenously,once a day for 3 consecutive times.After 6 months of infusion,PET/CT imaging was performed on the facial skin of young,elderly and elderly treatment groups,and the changes of SUVmax and CTmean were analyzed.Skin thickness,elasticity and water content were measured.HE staining was used to analyze the structural changes of the skin,Masson staining was used to compare the collagen fiber content in the skin,immunofluorescence staining was used to analyze the expression of P53,P21 and P16,and Tunel staining was used to analyze the apoptosis of the skin cells.3.Proteome analysis of macaque skin tissue:Mass spectrometry was used to capture proteome data of all groups of macaque monkey skin tissues.After mass spectrometry data was obtained,database search comparison and protein quantitative analysis were conducted through the Macaca proteome of Uniprot database.R analysis software was used to perform regression analysis on skin proteomic data of juvenile,young,middle and elderly macaques,and obtain protein molecules that changed significantly with age.The difference analysis of protein quantitative results between the senile group and the senile treatment group was carried out,and the intersection analysis of differential proteins and age-related proteins was conducted to obtain the"candidate molecules"related to reversing aging of BMSCs.4.Study on reversing HSF senescence by MSCs:（1）Establishment of HSFs senescence model:H2O2solution with final concentration of 0Um,100u M,200u M,300u M,400u M and500u M was prepared to induce HSFs for 24 h,48 h,72 h and 96 h,respectively.Real-time monitoring system was used to dynamically monitor the proliferation of HSFs,and SA-β-Gal staining was used to calculate the positive rate of senile cells.The cell cycle and apoptosis rate were analyzed by flow cytometry,and the optimal conditions of H2O2induction were obtained.（2）Establishment of h UCMSCs and senile HSFs co-culture system:h UCMSCs were co-cultured with senile HSFs for 72 h,and the HSFs were stained with SA-β-Gal and the positive rate of senile cells were counted.The migration force of HSFs was detected by cell migration assay,and the gene expression levels of p21 and p16 in HSFs were detected by q RT-PCR.The expression levels of P21,P16 andγ-H2AX in HSFs were analyzed by IF,and the ultrastructure of HSFs cells was observed by transmission electron microscopy.（3）q RT-PCR and Western Blot were used to verify candidate molecules and identify the key molecules of MSCs reversing skin aging.5.Functional verification of key molecules:RNAi technology was used to knock down the expression of key molecules in the fourth generation of HSFs,and OE technology was used to overexpress the key molecules in aging HSFs.For HSFs with knockout and overexpression of key molecules,the knockout and overexpression efficiency of HSFs were detected by q RT-PCR and Western Blot techniques,the SA-β-Gal activity of HSFs was detected by chemical chromogenic method,the gene expression levels of p21 and p16 were detected by q RT-PCR,and the intracellularγ-H2AX expression levels were detected by IF staining to clarify the function of key molecules.Results:1.The effect of BMSCs on skin aging of macaques:PET/CT imaging analysis showed that SUVmax and CTmean values of facial skin of the aged macaques were significantly lower than those of the young group（P<0.05）,and both were increased after BMSCs treatment,except for the significant difference in CTmean（P<0.01）.The elasticity and water content of skin tissue in the aged group were lower than those in the young group,but increased after BMSCs treatment（P<0.05）.The observation of tissue structure showed that,compared with the young group,the elderly group showed degenerative changes,which were improved after BMSCs treatment.Masson staining showed that the proportion of collagen fiber area in the skin of aged macaques was decreased compared with that in the young group（P<0.01）,and increased after BMSCs treatment,but the difference was not significant（P>0.05）.Immunofluorescence staining showed that the expression levels of P53,P21 and P16 were the highest in the elderly group,and the expression levels of P53 and P21 were significantly decreased after BMSCs treatment（P<0.05）.Tunel staining showed that the number of apoptotic cells in the aged group was the largest,and the number of apoptotic cells was significantly decreased after BMSCs treatment（P<0.05）.These results indicate that BMSCs can significantly improve the structure of aging skin of rhesus monkeys,and significantly reduce the expression level of aging marker molecules,playing a role in reversing skin aging.2.Results of skin tissue proteomics analysis:（1）Regression analysis showed that 224proteins showed significant changes in aging;The difference analysis of proteome data between the senile group and the senile treatment group showed 321 different proteins,among which 140 proteins showed increased expression and 181 proteins showed decreased expression after treatment.（2）Intersection analysis of differential proteins and proteins with significant changes in aging showed that the expression levels of 28 protein molecules were significantly reversed in the direction of aging after BMSCs treatment.The expression levels of 5 protein molecules decreased with the aging process and increased after BMSCs treatment,and the expression levels of 23 protein molecules increased with the aging process.BMSCs decreased after treatment.3.Effects of MSCs on aging skin fibroblasts:（1）Construction and evaluation of HSFs aging model:Cell dynamic monitoring showed that the proliferation of HSFs gradually decreased with the increase of H2O2concentration and time;When H2O2concentration was equal to 200μM,the positive rate of SA-β-Gal cells was the highest,and there was no significant difference between 72 h and 96 h（P>0.05）.When 200μM H2O2induced HSFs for72 h,the percentage of cells in G2/M phase increased（6.58%vs24.86%）,and the percentage of cells in S phase decreased（40.2%vs33.95%）,and the apoptosis rate increased significantly after induction（P<0.001）.（2）h UCMSCs and senile HSFs co-culture:after 72 h of h UCMSCs and senile HSFs co-culture,compared with the control group,the rate of SA-β-Gal positive cells and the gene expressions of p16 and p21 in the model group were significantly increased（P<0.01）,and significantly decreased（P<0.05）after co-culture.P21,P16 andγ-H2AX were highly expressed in the model group,and decreased after co-culture（P<0.01）.Compared with the control group,the number of cells passing through the filter membrane in the model group decreased（P<0.01）,and significantly increased after co-culture（P<0.01）.Transmission electron microscopy（TEM）observation showed that mitochondria of HSFs in the model group were swollen,less in number,cristae disappeared,and vacuolar changes.After co-culture,edema was reduced and mitochondria with normal structure could be seen.Compared with the control group,the expression level of HAT1 in the model group was significantly decreased（P<0.001）,and significantly increased after co-culture（P<0.01）.4.Functional verification of HAT1:（1）After HAT1 knockout by normal HSFs,the positive rate of SA-β-Gal in HSFs cells was significantly increased（P<0.001）,the gene expressions of p21 and p16 were significantly increased（P<0.05）,and the expression ofγ-H2AX was significantly increased（P<0.05）;（2）After HAT1 overexpression in aging HSFs,the positive rate of SA-β-Gal in HSFs cells was significantly decreased（P<0.05）,the gene expressions of p21 and p16 were significantly decreased（P<0.05）,andγ-H2AX was significantly decreased（P<0.05）.The results showed that knockdown of HAT1 could induce senescence of normal HSFs cells,and overexpression of HAT1 could significantly reduce the expression level of senescence marker molecules in senescent HSFs cells.HAT1 plays a key role in the regulation of skin cell senescence.Conslusions:1.BMSCs can significantly improve the skin tissue structure and reduce the expression level of age-related genes.2.The skin fibroblast senescence model was co-cultured with h UCMSCs,which proved that juvenile h UCMSCs could significantly reduce the expression of senescence marker genes,improve the ultrastructure of senescence cells and enhance the cell migration ability.3.Knockdown and overexpression studies have confirmed that HAT1 is a key gene closely related to aging... |
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