Effect of hypoxia treatment by CoCl2 on the expression of vascular growth factor in UC-MSCs

AIM: To study the effect of hypoxia induced by cobalt chloride (CoCl2) on the gene expression and protein secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in human umbilical cord mesenchymal stem cells (UC-MSCs). METHODS: UC-MSCs were isolated and cultivated. The third passage cells were cultured in medium containing 150 μmol/L CoCl2 for 72 h. The effect of hypoxia treatment on UC-MSCs surface markers (CD34, CD45, CD90, CD105, CD29 and CD49) was detected by flow cytometry. UC-MSCs were divided into four groups according to the concentration of CoCl2 (0 μmol/L, 50 μmol/L, 100 μmol/L and 150 μmol/L) and each group was divided into three subgroups according to three different times of hypoxia treatment (24, 48 and 72 h). mRNA and protein expression of bFGF and VEGF in UC-MSCs were examined by SYBRGREN fluorescent quantitation and ELISA. RESULTS: Hypoxia treatment with CoCl2 had no effect on cell morphology and surface markers. Gene expression of bFGF and VEGF enhanced with the increase of CoCl2 concentration at the same anaerobic cultivating time and reached its peak when the CoCl2 concentration was 150 μmol/L. When the CoCl2 concentration was 150 μmol/L, the gene expression of bFGF and VEGF also increased with the extension of hypoxia incubation time in the three subgroups, and they reached their peak at 48 h, respectively, 8.15±0.10 and 16.67±0.17. But gene expression decreased with the extension of hypoxia incubation time. Change in protein secretion was consistent with that in gene expression. Protein secretion of bFGF and VEGF also reached its peak at the CoCl2 concentration of 150 μmol/L after 48 h hypoxia-precondition, respectively, (69.63±7.90)ng/L and (89.55±5.45)ng/L. CONCLUSION: The biological function of UC-MSCs is only mildly affected by the appropriate concentration of CoCl2 (<150 μmol/L). CoCl2 can be used to stimulate hypoxic conditions. The effect of hypoxia on the secretion of bFGF and VEGF is time- and dose-dependent. The ideal conditions to promote the expression of bFGF, VEGF gene and protein are 150 μmol/L of CoCl2 and 48 h.