The Effects Of Hypoxia On The Chondrocytes Differentiation Ability Of Human Umbilical Cord-derived Mesenchymal Stem Cells

Objective The goal of this study was to master the method of isolation and culture of human umbilical cord-derived mesenchymal stem cells and to identify their biological characteristic; To explore the effects of hypoxia on the chondrocytes differentiation ability of the stem cells.Methods Isolation and culture the cells derived from the whartons jelly of the fetus umbilical cord in sterile by enzymatic treatment;then the digested cell suspensions were plated and cultured in DMEM/F12 medium containing10% FBS;While needing a cell growth to 80～90% fusion,1:3 spread generation culture. Observed the morphologic changes of the cells by the inverted microscope;The growth curve of the cells was made by MTT method. Meanwhile the immunophenotypic characterization of adherent cells were analyzed using flow cytometry. The third passage of the cells were inducated in the chondycytes inductive medium; According to the oxygen concentrations of the culture environmental the study was categorized into two groups:the control group (2%O2) and the exexperimental group (20%O2); After two weeks the cells were harvested between each grou and stained using toluidine blue and collagenⅡantibody; RT-PCR was used to detect the expression of HIF-1α,Sox-9 and CoL2al mRNA.Results Isolated cells grow adherence and the cells exhibited as long or short spindle-shape or multangular shape;The cells appeared almost fibroblast-like cells and cell adherence and proliferation were speeded up after passaged with 1:3 ratio; The growth curve made by MTT showed the P3,P5,P7 passage of the cells all had powerful proliferation ability; Flow cytometry shows that 96% and 95% of the P3 cells expressed CD44 and CD29 respectively, while only 1.8% of the cells expressed CD31 and 0.8% of the cells expressed CD45. After two weeks following chondrogenesis induction the experiment group show strong positive to toluidine blue stain and collagenⅡstain; However the control group exhibited a weaker reaction to the stain; The result of RT-PCR revealed the the experiment group expressed higher levels of the HIF-1α,Sox-9 and CoL2al mRNA than the control group and the difference was significantly(P<0.05)。Conclusion The stem cells derived from the whartons jelly of the human umbilical cord can be successful isolationed and cultured by enzymatic treatment;The hypoxia culture environmental can promote the chondrocytes differentiation of the HUMSC which may be related to the up-regulation of the HIF-1αmRNA expression.