Effect Of Autophagy On The Aging Injury Of Mice Bone Marrow Mesenchymal Stem Cells

Objective Bone marrow mesenchymal stem cells(BM-MSCs)were Isolated and cultured from young and old mice.The cell morphology,phenotype,apoptosis and autophagy were compared between the two groups to understand the relationship between autophagy and aging.Further signal pathway analysis was performed to clarify the detailed molecular mechanism of aging injury.Methods Clean grade healthy C57 mice were selected and divided into young group(8 weeks old)and old group(18 months old).The BM-MSCs were isolated from femur and tibia under sterile condition.Moreover,the third generation cells were used to carry out experiments.Cell morphology was observed under light microscope.The phenotype of two groups of BM-MSCs was detected by flow cytometry assay,Apoptosis was detected using TUNEL staining,then contrasting the distribution of apoptosis in two groups of BM-MSCs cells,and the staining results of apoptotic cells were observed by microscope.ELISA kit was used to detect the expressions of secretory protein VEGF,b FGF,IGF-1 and HGF in two groups of BM-MSCs cells.Western Botting assay was performed to detect the expressions of autophagy related protein LC3-I,LC3-II,Beclin 1,ATG12-5,p62 and signaling pathway related proteins,such as p-Akt?Akt?p-m TOR?m TOR.Results Both young and aged BM-MSCs were fibroblast-like morphology.Meanwhile,BM-MSCs in two groups were CD90 and CD29 positive,CD31 and CD45 negative.However,the apoptosis in aged BM-MSCs was significantly increased,compared with the young BM-MSCs(P<0.05).Meanwhile,the expressions of VEGF,b FGF,IGF-1 protein and HGF in aged BM-MSCs were significantly decreased than young BM-MSCs(P<0.05).Furthermore,in aged BM-MSCs,the expressions of autophagy signaling pathway related proteins,such as LC3-I/ LC3-II,Beclin 1 and ATG12-5,were decreased,whereas the P62 protein expression was increased(P<0.05).Moreover,the expressions of p-Akt and p-m TOR in aged BM-MSCs were increased than that in young BM-MSCs(P<0.05).Conclusions The cell morphology and phenotypes of aged BM-MSCs were are similar with those in young BM-MSCs.The apoptosis in aged BM-MSCs was increased than young BM-MSCs.Meanwhile,the paracrine function was impaired in aged BM-MSCs.However,the autophagy was decreased in aged BM-MSCs.Taken together,our results indicate that aging may down-regulated the autophagy by activating Akt/m TOR pathway,which may further lead to aging injury of BM-MSCs.