The Study Of Biological Characteristics Of Hypoxic Preconditioned Human Amniotic Mesenchymal Stem Cells

Objective:To study the effect of hypoxic preconditioning on the biologicalcharacteristics of human amniotic mesenchymal stem cells.Methods:(1)Isolating human amniotic membrane and extracting h AMSCs by mechanical method and enzymatic digestion method.(2)Pass the h AMSCs to the third generation and set hypoxic preconditioning group(3% O2,5% CO2)and normoxia group(21% O2,5%CO2).After 48 hours' preconditioning,observing the cell morphology under the inverted microscope.(3)Using immunocytochemistry and flow cytometry to identify the vimentin and immunophenotype of h AMSCs in hypoxic preconditioning group and normoxia group respectively.(4)The h AMSCs of hypoxic preconditioning group and normoxia group were osteogenic and adipogenic differentiation culture.After 3 weeks of induction,Evaluating the h AMSCs' condition of differentiation by alizarin red and oil red O staining.(5)Cell Counting Kit-8 and Edu cell proliferation assay was used to detect the proliferation activity and DNA synthesis rate of h AMSCs in hypoxic preconditioning group and normoxia group.(6)The h AMSCs of the hypoxic preconditioning group and normoxic group were starved for 24 hours in serum-free medium respectively.Detecting the Apoptosis of h AMSCs by Annexin V-FITC / PI flow cytometry.(7)The hypoxic preconditioning group and normoxia group h AMSCs were cultured in serum-free medium for 24 hours.Collecting the supernatant and using Proteome Profiler(ARY 007)kit to detect the related factor content which produced by secrete vascular in h AMSCs' supernatant.(8)The experimental data were statistically analyzed by SPSS 18.0 software.Results:(1)No morphological change was observed of hypoxia preconditioning and normoxic group h AMSCSs by inverted microscope observation.(2)h AMSCs vimentin expressed in both hypoxic preconditioning group and normoxiagroup,but without any detection in PBS control group.The expression of CD44,CD73,CD90 and CD105 in h AMSCs immunophenotype of both groups were highly expressed,CD19,CD34,CD45,CD11 b and HLA-DR are negative expressions,the difference between the two groups was was no statistical significance.(P> 0.05).(3)Both hypoxic preconditioning group and normoxia group human amniotic mesenchymal stem cells have osteogenic and adipogenic differentiation potentials.(4)The results of CCK-8 showed that the proliferation activity of amniotic mesenchymal stem cells in hypoxic preconditioning group and normoxia group first increased and then decreased from 1 to 7 days,and the proliferative ability in preconditioning group from 1 to7 was higher than that in normoxia group(P <0.01).Edu proliferation test results showed that DNA synthesis rate of preconditioning group increased significantly compared with normoxia group(P <0.05).(5)The FCM results of Annexin V-FITC/PI showed that the apoptosis of human amniotic mesenchymal stem cells in hypoxic preconditioning group(4.87±0.86%)was lower than that in normoxic group(12.3±1.14%).The difference was statistically significant(P < 0.01).(6)Proteome Profiler(ARY 007)results showed that hypoxic preconditioning group and h AMSCs cultured in serum-free medium,supernatant secreted 9 kinds of angiogenesisrelated growth factor:coagulation factor III,IGFBP,IL-8,MCP-1,Pentraxin E1,Serpin E1,Serpin F1,TIMP-1,Thrombospondin-2,and the supernatant of normoxic group can secrete angiogenesis-related growth factor: IGFBP,IL-8,MCP-1,pentraxin E1,Serpin E1,Serpin F1,TIMP-1,Thmbroospondin-2.Among them,the difference content of growth factor IGFBP,Pentraxin E1,Serpin E1 and Thrombospondin-2 between preconditioning group and normoxia group have statistical significance(P <0.05).Conclusions:3% hypoxic preconditioning can promote the proliferation,antiapoptosis and paracrine functions of human amniotic mesenchymal stem cells and maintain the characteristics of stem cells.