부산대학교 도서관

Objective To investigate the regulation effect of hypoxic/HIF-1α pathway on the differentiation of RAW264.7 cells into mature osteoclasts, and to explore the possible molecular mechanism. Methods RAW264.7 cell lines with stable low expression of HIF-1α and NRP-1 were respectively established by short hairpin RNA (shRNA) lentivirus infection. Then the RAW264.7 cells were cultured in groups, that is, control group, differentiation group [100 ng/mL RANKL+50% osteoblast conditioned medium (CM)], hypoxia group (100 ng/mL RANKL+50% osteoblast CM+100 μmol/L CoCl2), hypoxia+HIF-1α-NC group, hypoxia+HIF-1α shRNA group, hypoxia+NRP-1-NC group, and hypoxia+NRP-1-shRNA group. TRAP staining was used to observe osteoclast differentiation. The mRNA expression of Cath K, MMP-9, TRAP, HIF-1α and NRP-1 were detected by RT-PCR. Western blotting was used to detect the expression of HIF-1α and NRP-1 proteins. Results ① The mRNA expression levels of Cath K, MMP-9 and TRAP and the number of TRAP positive cells in the hypoxia groups were significantly lower than those in the differentiation groups (P < 0.05). ② The expression levels of HIF-1α and NRP-1 were significantly higher in the hypoxic groups than the differentiation groups (P < 0.05). ③ Compared with the hypoxia groups, the number of TRAP positive cells and the expression levels of osteoclast differentiation related genes were significantly increased, while those of HIF-1α and NRP-1 were significantly reduced in the hypoxic+HIF-1α shRNA group (P < 0.05). ④ Compared with the hypoxia groups, the number of TRAP positive cells and the expression levels of osteoclast differentiation related genes were significantly increased, while the level of NRP-1 was significantly decreased in the hypoxia+NRP-1-shRNA group (P < 0.05). Conclusion The hypoxia-mediated HIF-1α-dependent up-regulation of NRP-1 inhibits the differentiation of RAW264.7 cells into mature osteoclasts.