Study Of Mechanism Of Senescent Cell-Derived Extracellular Vesicles Regulate The Immune System

Cell senescence is a cellular state in which the cell cycle is permanently stagnant and can be induced by a variety of factors,the key of which is the imbalance of DNA damage.During individual aging process,the accumulation of senescent cells can be observed,but these two terms are not equivalent.Senescent cells have multiple functions such as inducing cell senescence of surrounding cells and conducting inflammatory response signaling pathways by releasing senescence associated secretory phenotype(SASP).In recent years,with the deepening of research in the field of molecular biology,extracellular vesicles(EVs)secreted by senescent cells are considered to play an important role in the aging-related microenvironment,and their functions and contents have attracted widespread attention.As vesicles that encapsulated by phospholipid bilayers secreted by all living cells,EVs exhibit heterogeneity in number,size,contents,and even production pathways in different cell states.So far,the regulatory function of EVs secreted by senescent fibroblasts on immune cells is still unclear.In this study,replicative senescent fibroblast-derived EVs(senescent cell derived EVs,SEN cells)and control young cell-derived EVs(Control cell derived EVs,CON EVs)were co-cultured with macrophages,and LC/MS was used to combine bioinformatics The analysis mines changes in protein differences in target cells and changes in cell signaling pathways.It was found that the NF-kB signaling pathway was activated after the uptake of SEN EVs by macrophages.To investigate the functional content of SEN EVs,we first knocked down miRNAs one by one in macrophages,and then co-cultured with SEN EVs.At the same time,real-time fluorescent quantitative PCR method was used to evaluate the expression levels of miRNAs in SEN and CON EVs.It was found that SEN EVs can specifically target the 3’TUTR region of SIRT1 through the high level of miR-30b-5p in them,thereby down-regulating the SIRT1 protein level in macrophages and activating the canonical NF-kB signaling pathway.At the same time,we detected the levels of EV-miR-30b-5p derived from the heart,lung,kidney and liver of naturally aging C57BL/6 mice,and found that only the miR-30b-5p level in liver EVs increased with age.In order to further study the specific mechanism of miR-30b-5p secreted into EVs in senescent cells,SAFB was found to be the key protein carrying miR-30b-5p into EVs by RNA pull down technology.However,the specific mechanism and function of SAFB secretion in EVs has not been reported yet.By isolating plasma EVs of mice of different ages,it was found that SAFB in plasma EVs of aging mice increased with age of mice.The mechanism is that SAFB in senescent cells accumulated in aging mice moves out of the nucleus,the activated Caspase 4 cuts the C-terminal,and finally the N-terminal of SAFB is sorted into multivesicles,and then released in the form of EVs.All in all,this thesis aims to study the function and mechanism of SEN EVs on immune cells in the aging related microenvironment.It is innovatively found that SAFB from aging fibroblasts can migrate out of the nucleus to assist miR-30b-5p to enter into EVs,and then down-regulate SIRT1 levels in macrophages and activate the canonical NF-κB signaling pathway.