The Role Of CD10 In The Regulation Of Heart Mesenchymal Stem Cell Senescence

Cellular senescence,a phenomenon in which cells permanently stop dividing and remain in a state of growth arrest,is a natural process that occurs as cells age and is thought to be a protective mechanism against damage caused by the replication of damaged or dysfunctional cells.Cellular senescence can be triggered by a variety of factors including DNA damage,telomere shortening,altered signalling through mitogenic and proliferation-related pathways,epigenetic changes and ectopic expression of tumour suppressors.When cells enter a senescent state,they typically undergo changes in morphology,gene expression and metabolism.The senescenceassociated secretory phenotypes refer to a number of cytokines,inflammatory factors and growth factors that are secreted by ageing cells.They are usually in response to senescence or other oxidative stress and plays a role in cell proliferation,inflammatory responses or angiogenesis.When presenting for a prolonged period,senescenceassociated secretory phenotypes can adversely affect the surrounding nuclear tissue,leading to reduced tissue functions,inflammatory responses or cell death.Senescenceassociated secretory phenotypes are therefore considered to be a phenomenon associated with ageing and many age-related diseases,including cancer,cardiovascular disease,diabetes and neurodegenerative diseases.Mesenchymal stem cells(MSCs)are a class of pluripotent stem cells that can differentiate into a variety of cell types including bone,cartilage,fat,muscle and connective tissue.They are found in various tissues of the body,including bone marrow,adipose tissue,umbilical cords and dental pulp,and are important for tissue repair and regeneration,immune regulation and cellular ageing.MSCs have been shown to play a role in improving patients’ spinal cord injuries or reducing cardiovascular disease.However,as we getting older,the ability of MSCs to renew themselves declines and senescence occurs,during which time MSCs undergo morphological changes.CD 10 is a neutral endopeptidase that can be expressed in a variety of stem cells.Heart is the major source of soluble circulating CD 10 in the body.CD 10 inhibitors have been shown to reduce cardiac damage in the treatment of cardiovascular disease.However,the relationship between CD 10 and stem cell senescence is seldom reported.In the present study,we found CD 10 expression level and activity changed in the senescent heart MSCs.However,how CD 10 regulates stem cell senescence and the underlying molecular mechanisms are unclear.Therefore,in this study,we established a HMSC senescence model in which CD 10 was knocked down and overexpressed to investigate the role of CD 10 in the process of HMSC senescence.Our aim is to provide new ideas for delaying cardiovascular senescence.Methods1.For the establishment of an in vitro model of senescence,cellular senescence was detected by β-gal cell senescence staining;telomere length changes and expression of senescence-related molecules were detected by agarose gel electrophoresis and quantitative fluorescence PCR(qRT-PCR).2.The changes in cell signalling pathways and senescence-associated factors were detected by transcriptome sequencing;CD 10 expression in cardiac mesenchymal stem cells(HMSC)was detected by western blot,qRT-PCR and immunofluorescence.3.CD 10 was overexpressed by using lentiviral transfection,and the effect of CD 10 on the expression of senescence-related factors was investigated by western blot,qRTPCR and β-gal cellular senescence staining.4.The effect of thiorphan,a CD 10 inhibitor,on HMSC proliferation and senescence was detected by MTT and β-gal cell senescence staining;the effect of thiorphan on PTEN-AKT pathway and the expression of senescence-related factors were detected by western blot.5.In the CD 10 knocking down HMSC cells,we used MTT to detect the changes of cell proliferation,flow cytometry to detect changes in cell cycle,western blot to detect changes in cell cycle-related and apoptosis-related proteins.Results1.When the HMSCs cells were continuous passaged,the cellular morphology changed,including cellular size increase,borders becoming blurred,β-gal staining increase,telomere length shortening,expression of senescence-associated molecules p16 and p27 upregulated,and expression of p53 and WNT5A decrease.2.In the senescent HMSCs,the CD 10 expression level were increased,the PTENAkt signalling pathway was activated,and the expression level of the cell cycle-related proteinswere downregualted.3.CD 10 overexpression accelerated the HMSCs senescence,activated the PTENAkt signalling pathway and upregulated the expression of senescence-associated molecules p16 and p27.4.A high concentration of Thi(4.0 mM)inhibited HMSC proliferation,while a low concentration of Thi(0.1 mM)promoted HMSC proliferation and delayed cellular senescence;PTEN-Akt signalling pathway was inhibited after the addition of 0.1 mM Thi,and p16 and p27 expression was also inhibited.5.CD 10 knockdown decreased cell proliferation,induced G2/M cell cycle arrest,decreased the expression level of cyclin B1,cyclin D1,CDK1,CDK2 and CDK4,and increased the expression level of caspase3,caspase7 and p21.Conclusions1.Senescence occurs with continuous passage of HMSCs.2.CD 10 level increases in senescent HMSC.3.Low concentration of Thi attenuates CD 10-induced cellular senescence.4.CD 10 has a dual role in the HMSC senescence:low level of CD 10 slows down HMSC senescence and high levels of CD 10 promote HMSC senescence.