Effects Of SIRT On Hypoxia-induced Proliferation Of Human Umbilical Cord-derived Mesenchymal Stem Cells

Background:Mesenchymal stem cells(MSCs)are a type of pluripotent stem cell originated from early mesoderm.It has self-renewal and differentiation capacity into many lineages such as adipocytes,osteoblasts,chondrocytes and so on in vitro.They are an important cell source in tissue engineering and regenerative medicine.There are many kinds of mesenchymal stem cells in stem cell research filed,such as bone marrow mesenchymal stem cells,adipose mesenchymal stem cells,skeletal muscle mesenchymal stem cells and so on.However,Human umbilical cord mesenchymal stem cells(hUC-MSCs)due to their many advantages like easy isolation,low immunogenicity,a broad of source,less bioethics issues and better proliferation ability becoming a more appropriate source of mesenchymal stem cells gradually.But,when mesenchymal stem cells transplanted into human body,a lot of mesenchymal stem cells easily apoptosis or die because of lacking of nutrition,ischemia or hypoxia in the engrafted environment.In addition,with the expansion of hUC-MSCs,the proliferation rate is slowed down,the gene is unstable,the stem cell characteristic is gradually missing,and the aging process is started etc.In normoxia condition,these disadvantages greatly limits the wide application in clinical practice.The concentrations of oxygen in human tissues ranges from 3-11%,while the regions where stem cells exists are mostly hypoxic.1-9%O2,veritably mimic physiological hypoxic environments in vivo.Lots of studies have shown that hypoxia plays an important role in enhancing the ability of proliferation and differentiation of stem cells.The stem cells preconditioned with hypoxia condition have stronger tissue repair capacity.Therefore,the application of hypoxia in stem cells has been interested.Sirtuin is one type of epigenetic regulation proteins with the abilities of catalyze both deacetylation and ADP-ribosylation.The mammalian sirtuin family contains seven proteins,SIRT1-SIRT7,which are found to be involved in cell proliferation,senescence,apoptosis,oxidative stress and metabolism regulation,extended cell life and other biological processes.However,the effect of hypoxia on the sirtuin protein of mesenchymal stem cells is still confused,so,this study aims investigate whether the localization of SIRT1,2,3,4,5,6 in hUC-MSCs changed under hypoxia condition(5%O2)and the effects of hypoxia on the expression of SIRT1 protein in the nucleus protein SIRT1,cytoplasmic protein SIRT2 and mitochondrial SIRT5 protein respectively.With the purpose of providing evidence for further exploring the role of sirtuin in hypoxia induced hUC-MSCs proliferation.Methods1,Human umbilical cord mesenchymal stem cells were isolated by the explant technique method,we identified the cells isolated by adherent method from the following three aspects:?The cell morphology under inverted microscope.?The surface marker CD90,CD105,CD44,CD29,CD14,CD14,CD19,CD34,CD45 and HLA-DR3.?The ability of adipose and osteogenic differentiation.2,The proliferation of hUC-MSCs under normoxic(21%O2)and hypoxic(5%O2)conditions was measured from three aspects.?Growth curve,hUC-MSCs were placed under hypoxia and normoxia for 7 days.?CCK-8 assy for detection of hUC-MSCs' proliferation in hypoxia and normoxia.?Cell cycle was analyzed by flow cytometry.3,Immunofluorescence assay was used to detect the location of SIRT(1-6)undernomoxia and hypoxia conditions.Expressions of SIRT(1,2,5)under normoxia and hypoxia conditions for different times(24h,42h,72h)were detected by Western blot.Results1,We successfully isolated hUC-MSCs from human umbilical cord.The cells were characteristic of fibroblast-like morphology on the flask.Flow cytometry showed that hUC-MSCs were CD105,CD90,CD44 and CD29 positive,but CD14,CD19,CD34,CD45 and HLA-DR negative.The isolated cells showed multilineage differentiation potential under certain conditions.2,The growth curve of hUC-MSCs cultured in hypoxia was higher than normoxic.CCK-8 showed that proliferation of hUC-MSCs was enhanced under hypoxia compared with normoxia(Pall<0.05).Cell cycle was analyzed by flow cytometry indicated that the ratio of S phase cells was higher than that of normoxic culture for 48 h(Pall<0.05)3,Immunofluorescence showed that no matter in hypoxia or nomoxia conditions,SIRT1 and 6 were mainly localized in the nucleus,SIRT2 was mainly localized in cytoplasm,and SIRT(3,4,5)were localized in mitochondria.Western blot showed the expression of SIRT1 in hUC-MSCs increased under hypoxia after being cultured for 24 h and 48 h(Pall<0.05).Similarly,the expression of SIRT5 was increased under hypoxia at three times(Pall<0.05).The expression of SIRT2 in hypoxia was increased after being cultured 24 h and 72 h compared with normoxic cells(Pall<0.05),however,the hypoxic cells' expression of SIRT2 was lower than normoxic cells' after being cultured 48 h(P<0.05).Conclusions1,Hypoxia can increase hUC-MSCs' proliferation.2,Expessions of SIRT(1,2,5)were affected by hypoxia,which implies that SIRT(1,2,5)may be involved in the regulation of hypoxia induced proliferation of hUC-MSCs.