Effects Of Hypoxia On The Biological Behaviors And Paracrine Functions Of Mesenchymal Stem Cells

Mesenchymal stem cells?MSCs?possessed the ability of self-renewal and can differentiate into variety cells,furthermore they involved in regulated the immune response.Above all they are regarded as promising cell-based tool in tissue repair and regeneration medicine.However low survival and restricted migrated to lesion tissue after injected into the recipient limited the widely application.Thus the investigations to improve the MSCs are in need.Generally MSCs cultured under 21%O₂ while under physiological conditions in vivo MSCs exposed to the hypoxia niche that the oxygen concentration vary between 1%to 7%.What are the effects of different oxygen concentrations on the biological functions of MSCs?In this research we study the hypoxia culture on the biochracteristics and paracrine functions of bone marrow derived mesenchymal stem cells?BMSCs?.Part 1Effects of different culture conditions on the biocharacteristics of BMSCs.Objective:Compare the influence of different oxygen concentration on the cell morphology,immunophenotype,proliferation,migration,adhesion and adhesive molecules level of BMSCs.Methods:BMSCs were obtained from SD?Sprague-Dawley?rat and then cultured in1%O₂,94%N₂,5%CO₂,37?incubator and 21%O₂,5%CO₂,37?incubator.Detected the cell morphology,proliferation level,and also migrate and adhesive properties of BMSCs.Results:Different culture condition makes no difference on cell morphology and cell immunophenotype;while hypoxia cultured BMSCs expanded with low proliferation;Moreover compared to normoxia group,cells under hypoxia appeared enhanced migration property at 16 h and 24 h?P<0.05?;And the numbers of adherent cells were increased under hypoxia compared to normoxia condition at 1 h and 3 h?P<0.001?.At last,hypoxia culture condition inhibited the expression of CD29 at P1?P<0.05?while promoted the expression at P2?P<0.05?.However there are no significant effects on CD11b.Conclusions:1%O₂ culture condition inhibited cell proliferation,while enhanced the ability of migration and adhesion.Part 2 Effects of different culture conditions on the paracrine functions of BMSCs.Objective: Fibroblast?L929?and human brain microvascular endothelial cells?HBMECs?were used as the major subjects to test the paracrine function of BMSCs.Methods:1.BMSCs were cultured in normoxia condition,hypoxia condition and hypoxia pretreatment condition and then collected the cultured conditioned medium accordingly;Normoxia cultured conditioned medium obtained from cells cultured in21%O2,Hypoxia cultured conditioned medium obtained from cells cultured in 1%O2,while transfer the 21%O2cultured BMSCs to 1%O2for 24 h before collected the conditioned medium for hypoxia pretreatment conditioned medium.2.L929 were cocultured with the conditioned medium described above and detect the cell proliferation,cell viability after oxidative stress injury that treated with H2O2 and migration function through CCK-8 and scratch assay.And cocultured HBMECs with conditioned medium and test the effect of tube formation through tube formation assay.3.Detect the secretion level of VEGF,PDGF,b FGF in different conditioned medium from BMSCs by ELISA kit.Results:1.After cocultured 48 h the proliferation of L929 showed significantly increased in different conditioned mediums,while no remarkably difference between three experimental groups.While at 72 h only hypoxia pretreatment cultured conditioned medium group indicated higher proliferation level compared with normoxia cultured conditioned medium?P<0.05?group and hypoxia cultured conditioned medium group?P<0.01?.H2O2 decreased the cell viability of L929 significantly,and hypoxia pretreatment conditioned medium improved the cell injury of L929?P<0.01?.While there are no significant effects when treated with normoxia cultured and hypoxia cultured conditioned medium.2.L929 were treated 12 h with conditioned medium,and it showed that at 12 h the migration level were higher than routine cultured L929?P<0.01?but no significant difference between the three different conditioned medium.3.Hypoxia pretreatment conditioned medium make no significant difference on the tube formation ability of HBMECs when compared with control group,while another two conditioned mediums inhibit the tube formation functions.4.The VEGF and PDGF level from different cultured supernatant have no significant difference,while the secretion of b FGF were higher in hypoxia pretreatment conditioned medium than normoxia cultured conditioned medium?P<0.05?and hypoxia cultured conditioned medium?P<0.01?.Conclusions: Hypoxia pretreatment cultured conditioned medium increased the proliferation of L929,improved the oxidative stress induced injury and increased the secretion level of b FGF when compared with normoxia cultured conditioned medium and hypoxia cultured conditioned medium.