| ObjectiveIn this study,Rat TSPCs were isolated from 6-month-old and 24-month-old male Sprague-Dawley rat are the main research object,aiming to investigate the FOXP1 expression pattern in young and aged TSPCs in vitro and explore the role of FOXP1 in age-related dysfunction,such as self-renewal,migration and differentiation,and its in-depth mechanism was also investigated.Methods1.Rat TSPCs were isolated from 6-month-old and 24-month-old male Sprague-Dawley.Clone-forming ability of TSPCs was detected by clone formation assay.The surface antigen expressions of TSPCs were measured by flow cytometry.Oil red staining,Alizarin red staining and Alican staining were used to detect the adipogenic,osteogenic and chondrogenic differentiation potential of TSPCs.2.Cellular senescence was examined by β-gal staining,FOXP1 and p161NK4 expressions in TSPCs were investigated by Western blot and Quantitative real time polymerase chain reaction(qRT-PCR).3.The lentiviral vectors mediated FOXP1(LV-FOXP1)was used to transfect the aged TSPCs,FOXP1-siRNA was used to transfect the young TSPCs.The transfect efficiency of the LV-FOXP1 was explored by Western blot.Cellular senescence in different groups were examined by β-gal staining.p16INK4 expression was detected by Western blot.self-renewal ability of TSPCs was examined by clone formation assay and CCK8 assay.To investigate migration of TSPCs,we used an in vitro scratch assay in confluent LV-FOXP1 cultures.we investigated the expression of key tendon-related markers(TNMD,SCX and Collal)of TSPCs.4.The expressions of cell cycle-rated proteins(E2F1,pRb and Cyclin D1)were examined by Western blot.Results1.The rat TSPCs showed fibroblast-like morphology and colonies were observed after 7 days in culture.TSPCs expressed MSC markers CD44 and CD90.1.and were negative for hematopoietic marker CD34,leukocyte marker CD45,and endothelial cell marker CD31.Lipid droplets were formed after incubating the TSPCs in adipogenic medium for 15 days.Osteogenic differentiation ability was detected by Alizarin Red staining,as the mineralized calcium deposits were formed after 20 days of induction.The chondrogenic differentiation of TSPCs was proved by positive Alican staining after 20 days of micromass culture and induction.2.Cellular senescence was examined by(3-gal staining,The number of β-gal-positive cells was significantly increased in aged cells.Meanwhile,the senescence marker p’IWK4A protein level of aged TSPCs was markedly increased.The expression patterns of FOXP1 in TSPCs were examined by qRT-PCR and Western blot.Compared with young TSPCs,FOXP1 mRNA and protein levels were markedly decreased in the aged TSPCs.3.Aged cells were transduced with LV-FOXP1 to overexpress FOXP1,we explored the efficiency of the LV-FOXP1 by Western blot and analysis showed that LV-FOXP1 increased the protein level of FOXP1 compared with control in aged TSPCs.β-gal staining demonstrated that FOXP1 overexpression reduced the number of β-gal positive cells.Western blot also showed p16INK4A expression were decreased after LV-FOXPl transduction.We used FOXP1-siRNA to knockdown FOXP1 in young TSPCs,β-gal staining showed that FOXP1-siRNA increased the percentage of p-gal positive cells.Moreover,FOXP1 deletion also increased the expression of p16INK4A in young TSPCs.Clone formation assay assays showed a significantly higher colony number in the LV-FOXP1 group.To further verify the observed self-renewal change,we analyzed the proliferative activity of the TSPCs by CCK-8 assay.We used an in vitro scratch assay to detected the migration ability,and LV-FOXP1 treatment promoted aged TSPCs migration.The result demonstrated that FOXP1 overexpression significantly increased the proliferative rate compared with control.We investigated the expression of key tendon-related markers of TSPCs,the results of the qRT-PCR assay showed that the mRNA levels of Scx,Tnmd and Collal were reduced in aged TSPCs.LV-FOXP1 treatment significantly increased the expression of tendon-related markers in aged TSPCs.4.We examined the expression of cell cycle-rated proteins by Western blot.The result indicated that E2F1,pRb and cyclin D1 expression were decreased in aged TSPCs.Moreover,FOXP1 overexpression notably increased these cell cycle-rated proteins levels compared with young TSPCs.Conclusion1.The expression of FOXP1 in aged TSPCs was significantly decreased,FOXP1 might be associated with TSPCs aging.2.FOXP1 overexpression attenuates TSPCs aging and restores the age-associated reduction of self-renewal,migration and differentiation.FOXP1 may be a potential molecule target for antagonizing tendon aging.3.FOXP1 could present a specific inhibitory effect on TSPCs aging via modulated cell cycle progression. |
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