[Hypoxia regulates the expression of OPG/RANKL mRNA in rat bone marrow mesenchymal stem cells].

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 CoCl (cobalt chloride). rBMSCs were incubated in cell culture mediums with different concentrations of CoCl (final concentrations of CoCl were 0, 50, 100, 200, 400 μmol/L) and incubated for different times. MTT assay was applied to detect the effect of CoCl 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 CoCl 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 CoCl inhibited the proliferation of rBMSCs (P<0.05). However, 50, 100 μmol/L CoCl 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 CoCl groups was significantly higher than the control group; the effect of 100 μmol/L CoCl was significantly greater than 50 μmol/L CoCl. 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 (P<0.05).

CONCLUSIONS

Hypoxia can regulate the mRNA expression of OPG and RANKL mRNA in rBMSCs and significantly promote osteogenic differentiation.