Abstract: PURPOSE: To investigate the effects of hypoxia on the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) mRNA in rat bone marrow mesenchymal stem cells (rBMSCs). METHODS: rBMSCs were isolated and cultured by whole bone marrow cell adherent method, and an optimal hypoxic preconditioning model was established with CoCl₂ (cobalt chloride). rBMSCs were incubated in cell culture mediums with different concentrations of CoCl₂ (final concentrations of CoCl₂ were 0, 50, 100, 200, 400 μmol/L) and incubated for different times. MTT assay was applied to detect the effect of CoCl₂ on cell proliferation. mRNA and protein expression of HIF-1α of rBMSCs was detected by real-time PCR and Western blot. After treated with 100 μmol/L CoCl₂ for 0, 12, 24, 48, 72, 96 h, the expression of rBMSCs OPG/RANKL mRNA were detected by real-time PCR. The differences in distribution of each genotype were analyzed with SPSS 18.0 software package. RESULTS: Compared with the control group, 200, 400 μmol/L CoCl₂ inhibited the proliferation of rBMSCs (P<0.05). However, 50, 100 μmol/L CoCl₂ had no significant impact on the proliferation of rBMSCs (P>0.05). Real-time PCR and Western blot showed that HIF-1α expression in 50 μmol/L and 100 μmol/L CoCl₂ groups was significantly higher than the control group; the effect of 100 μmol/L CoCl₂ was significantly greater than 50 μmol/L CoCl₂. After cultivated in hypoxia condition for 12 h, the expression of OPG and RANKL mRNA in rBMSCs didn't change significantly (P>0.05). After cultured hypoxia condition for 24, 48, 72, 96 h, the expression of OPG mRNA in rBMSCs increased while the RANKL decreased, thus the ratio of OPG/RANKL increased and the difference was significant

Key words: Rat bone marrow derived mesenchymal stem cells, Hypoxia, Osteoprotegerin, Receptor activator of NF-κb ligand