Exploration And Analysis Of The Correlation Between Wnt2 Protein And Senescence Of Human Mesenchymal Stem Cells In Vitro

Objective:This study aimed to explore the expression and biological function of the Wnt family members in hMSCs by analyzing relationship between the canonical Wnt/?-catenin signaling pathway and the proliferation of hMSCs in human mesenchymal stem cells,and to find a biological marker that can represent hMSCs replicative aging in hMSCs in vitro,and to provide new technical strategies for hMSCs quality evaluation.Methods and Results:Real Time-PCR was used to detect the transcriptional level of various Wnt genes in hMSCs at different passage numbers from different sources,trying to find the relationship between the expression level of Wnt genes and replicative senescence,especially the involvement of the Wnt2 gene in replicative senescence of the hMSCs different passage numbers.The results showed that the expression of Wnt2 gene was generally higher than that of other Wnt family members,and the transcription level of Wnt2 subtypes decreased with the increase of passage numbers in the standard hMSCs cell lines from different sources.In addition,the hMSCs from different sources through Western blot and ELISA showed that the expression level of Wnt2 protein and the standard strain decreased with the increase of passage mumbers,and this was also verified through both Real Time-PCR analysis and absolute quantitative PCR analysis of Wnt2 gene.In this study,to determine whether Wnt2 can activate canonical signaling pathway through the luciferase reporter assay by determining activity of the downstream target gene TCF1 promoter and nuclear translocation of the?-catenin protein through the Wnt/?-catenin signaling pathway.We found that Wnt2 protein could activate the downstream target gene TCF1 of canonical signaling pathway,and can induce the accumulation of?-catenin into the nucleus.Exogenous Wnt2 recombinant protein can activate distal downstream WISP1,which proves that the possibility of canonical Wnt2/?-catenin overactivation induced degenerative disease.Meanwhile,the correlation between Wnt2 and hMSCs replicative senescence was further explored by using the hMSCs senescence model induced by H2O2,and the relationship between GSK-3 beta and Wnt2 expression was determined by H₂O₂ aging model and GSK-3 beta inhibitor CHIR99021.In the hMSCs senescence model induced by H₂O₂,the expression of Wnt2 decreased with the concentration of H₂O₂,and the expression of GSK-3?was increased with the senescence expression;the enhancement of GSK-3?level further inhibited Wnt signaling transduction;supplementation of GSK-3?inhibitor CHIR99021 promoted the level of Wnt2 and activated the Wnt canonical signals to a certain extent.Conclusion:In conclusion,the study confirmed that Wnt2 was a key Wnt molecule and onset associated with hMSCs replication and senescence in the canonical Wnt/?-catenin signaling pathway.The change of its expression level can be used to judge the cell replicative senescence,so it can be used as a new molecular marker for cell reproduction and senescence.At the same time,the detection of Wnt2 expression level can be used as a new method for hMSCs quality evaluation.