Effects Of Hypoxia On Proliferation And Pluri Potency Maintenance Of Buffalo Adipose-derived Stem Cells

The purpose of this research was to investigate the effects of hypoxia on proliferation and biological characteristics maintenance of buffalo adipose-derived stem cells in vitro.bASCs were digested from buffalo neck adipose tissue with type ? collagenase.And then,cell morphology,growth curve,cell colony formation ability,relative expression levels of proliferation and apoptosis related genes and secretion of growth factors bFGF,VEGF of hypoxia?5%O₂?and normoxia?20%O₂?cultured bASCs were analysed seperately.Meanwhile,expression of MSCs markers and pluripotency genes in serially passaged hypoxia and,normoxia bASCs were also analysed.And the interaction mechanism of HIF-1 pathway,bFGF and VEGF on bASCs proliferation in hypoxia culture was also explored.The results were shown as following.?1?0.1%Roche type ? collagenase deteched bASCs begun to adhere the culture dishes after 4h of seeding.While 48h later,bASCs presented as fibroblast-like shuttle or polygon,and growed as helix under inverted microscope.Hypoxia-cultured bASCs presented as stereoscopic shuttle in the course of serially passaged,while bASCs presented as loose and vacuolus after serially passaged to P9 in normoxia.Meanwhlie,cell proliferation rate and cell colony number of hypoxia-cultured bASCs were significantly higher than normoxia-cultured bASCs?P<0.05?.Hypoxia significantly up-regulated proliferation gene KI-67,anti apoptotic gene Bcl-2,growth factors genes:bFGF,VEGFR-1?P<0.05?,while significantly down-regulated pro-apoptotic genes:Caspase-3 and Bax?P<0.05?.And hypoxia significantly promoted bASCs secreted growth factors bFGF and VEGF?P<0.05?.?2?Inhibited bFGF related pathways in hypoxia environments leaded to suppression of cell proliferation,down-regulation of HIF-1? and reduction of bFGF,VEGF secretion;while inhibited HIF-1? expression could also resulted in suppression of cell proliferationthe,down-regulation of bFGF and VEGF and reduction of bFGF,VEGF secretion;inhibited HCF-1? and added bFGF,VEGF at the same time could counteract the side effect of inhibitting HIF-1? on bASCs proliferation.?3?bASCs expressed pluripotency genes:OCT4,SOX2,C-MYC,NANOG,KLF4 and MSCs surface marker antigens:CD44,CD60,CD73,CD90 and CD 105.While didn't express haematopoietic marker CD45 in hypoxia and normoxia.Hypoxia significantly up-regulated pluripotency genes:OCT4,NANOG and C-MYC?P<0.05?Hypoxia significantly up-regulated adipocyte,osteocyte and chondrocyte specific genes PPAR-?,RUNX2 and COL2A1?P<0.05?.What's more,hypoxia-cultured bASCs also exhibited significantly higher positive rate of neurocyte specific genes TUBB3 than normoxia-culture bASCs?P<0.05?.On the whole,type ? collagenase is able to detach bASCs,which with typical biological characteristics of MSCs.5%hypoxia promotes bASCs proliferation and decreases apoptosis through interaction of HIF-1?,bFGF and VEGF;hypoxia environment is more favorable to bASCs pluripotency maintenance and multiple differentiation potential improvement.