Effect Of Hypoxia-preconditioning On Expression Of Vascular Growth Factor In UC-MSCs

Objective To explore the isolation and cultivation method of human umbilical cord derived mesenchymal stem cells and to research the effect of cobalt chloride (CoCl2) hypoxia on their biological characteristics.To investigate the influence of hypoxia-preconditioning on the gene expression and protein secretion of bFGF and VEGF.Method The umbilical cord from full-term section patients were collected in sterile condition immediately upon delivery. Blood clot in umbilical arteries and veins were rinsed out by PBS,The umbilical cord was then cut into pieces of 1mm3.UC were incubated with collagenase typeⅣ(0.1%) for 60 min and then trypsin(0.125%) for 30min with gentle agitation at 37℃.The digested mixture was then passed through a filter to obtain cell suspension. Cells were plated in cell culture flask in DMEM (5% FBS)medium.Once 80-90% confluence had been reached,adherent cells were replated at 1:3 under the same culture conditions to amplify. Morphological characteristic of the cells has been observed by phase contrast microscope and the expression of cell surface markers was detected by flow cytometry (CD34N,CD45,CD,CD105,CD29及CD49).The 3rd passages of UC-MSCs were conventionally cultured. The effect of CoCl2 (0,50,100,150,200,250μmol/L) on vitality and proliferation of UC-MSCs was detected by MTT assay. Detected the surface marker of UC-MSCs after hypoxia pretreatment 72h, with the culture juice containing 150μmol/L CoCl2.The UC-MSCs were divided into four groups according to the concentration of CoCl2:control, low, middle and high group.Each group was treated for 24h,48h and 72h. The expression of bFGF,VEGF mRNA were detected with fluorescent quantitation RT-PCR, the protein expression of bFGF and VEGF were detected with ELLSA.Results mesenchycal stem cells were isolated from UC by collagenase and trypsin digestion sufficiently. Adherent cells were observed after 24 hours, the cells demonstrated a fibroblast-like phenotype. Training 4～5d, the adherent cells were increased, cell demonstrated spindle or irregular phenotype, and reached approximately 80～90% confluence after 8～9days, Passage once a week,No obvious morphological changes in before and after cell passage. The control group,Cell growth curve showed "S"-shaped, After CoCl2 treatment, cell growth curve of each group were irregular "S"-shaped, early logarithmic phase, After hypoxia, cell proliferation were accelerated, ogarithmic phase shorter than the control, After 3 days, the cell proliferation slowed,,into the platform,4 to 5 days, the cell proliferation were decreased, the platform of no significant. When CoCl2 higher concentration (200,250μmol/L), the cell growth curve tends to low and flat. The UC-MSCs were still highly expressed CD90, CD29,CD49 and CD 105,low expression of CD45 and CD34, after hypoxic preconditioning by CoCl2,No obvious morphological changes in before and behind hypoxic preconditioning by CoCl2.the bFGF,VEGF gene have time and dose dependence The bFGF was significantly higher in three CoCl2 groups(50,100,150μmol/L) than in control group(0μmol/L)(P<0.05),The mRNA expression of bFGF,no significant differences were observed between.24h,48h, and. 72 h time point, after 100μmol/L CoCl2 treatment, the expression level of mRNA increases gradually with the time.After 150μmol/L CoCl2 treatment, the 48h mRNA is obviously higher than 24h, but 72h is lower than 48h. The VEGF was significantly higher in three CoCl2groups(50,100,150μmol/L) than in control group(0μmol/L) (P<0.05),After 50μmol/L CoCl2 treatment, there are no obvious differences.After 100μmol/L and 150μmol/L CoCl2 treatment, the mRNA gradually increase with the time(P<0.05),and all reach peak at 48h.150μmol/L group is obviously higher than 100μmol/L group, the discrepancy has statistical significance(P<0.05).The protein expression of bFGF,VEGF are coincidence to the gene.CONCLUSION Cobalt chloride in Proper concentration(=150μmol/L) can simulate cell hypoxia because of its little influence on the reproductive activity of UC-MSCs,Hypoxia-preconditioning can promote the secretion of the bFGF,VEGF from the UC-MSCs,The 150μmol/L cobalt chloride 48h hypoxia-preconditioning is the optimum condition in which it can promote the protein secretion and gene expression of bFGF, VEGF.