低氧状态下骨细胞参与破骨细胞形成的作用机制研究

Mechanism of participation of osteocytes in the formation of osteoclasts under hypoxia

目的探究低氧状态下骨细胞对破骨细胞形成的作用及其机制。方法用去铁胺甲磺酸(DFO)刺激骨细胞系MLO-Y4建立体外低氧骨细胞培养体系。CCK-8实验检测DFO对MLO-Y4细胞增殖活性的影响;采用DFO处理后的MLO-Y4细胞培养液制备条件培养基诱导RAW264.7细胞分化,行抗酒石酸酸性磷酸酶(TRAP)染色检测;利用实时荧光定量聚合酶链反应、细胞免疫荧光和蛋白质印迹(Western blot)检测DFO作用下MLO-Y4表达低氧诱导因子(HIF)-1α与核因子κB受体活化因子配体(RANKL)的情况;检测HIF-1α siRNA转染对MLO-Y4表达HIF-1α和RANKL的影响。结果 100 μmol·L^-1 DFO作用24 h时MLO-Y4增殖活性升高,之后随着时间延长细胞增殖活性下降(P<0.01)。加入可溶性RANKL后,低氧组可见破骨细胞形成明显增加(P<0.001)。100 μmol·L^-1 DFO作用下,HIF-1α mRNA表达稳定,RANKL mRNA的表达随时间明显变化,24 h时最高(P<0.01);HIF-1α、RANKL蛋白表达升高(P<0.01)。低氧状态下siHIF-1α转染可降低HIF-1α和RANKL的表达(P<0.01),破骨细胞减少(P<0.01)。结论低氧状态下MLO-Y4可通过上调HIF-1α的蛋白水平促进RANKL的生成,进而加速RAW264.7细胞向破骨细胞分化。

Objective To investigate the mechanism of the participation of osteocytes in the formation of osteoclasts under hypoxia. Methods The hypoxia culture system of osteocyte-like cell line MLO-Y4 was established by deferoxamine mesylate (DFO) in vitro. The proliferation of MLO-Y4 cells was examined by CCK-8 cell proliferation/toxicity assay. RAW264.7 cells were induced to osteoclasts by the conditioned medium containing the cultured MLO-Y4. Tartrate-resistant acid phosphatase (TRAP) staining was performed on day 7. Quantitative real-time fluorescence polymerase chain reaction, immunofluorescence, and Western blot were used to detect the expression levels of hypoxia-inducible factor (HIF)-1α and receptor activator of nuclear factor-κB ligand (RANKL) in MLO-Y4 under hypoxia. The effects of siHIF-1α on the expression levels of HIF-1α and RANKL in MLO-Y4 under the same conditions were detected. Results DFO (100 μmol·L^-1) promoted the proliferation of MLO-Y4 at 24 h, which decreased with time (P<0.01). After the addition of soluble sRANKL, the formation of osteoclasts was significantly increased in the DFO group (P<0.001). The expression of RANKL mRNA in MLO-Y4 under 100 μmol·L^-1 DFO increased first and then decreased with the duration of hypoxia. This expression reached a peak at 24 h (P<0.01). Hypoxia up-regulated the expression of HIF-1α and RANKL protein (P<0.01). Under hypoxia, siHIF-1α downregulated the expression of HIF-1α and RANKL (P<0.01). siHIF-1α also decreased the number of osteoclasts (P<0.01). Conclusion Under hypoxia, MLO-Y4 could facilitate the formation of RANKL through upregulating the expression of HIF-1α protein, thereby accelerate the differentiation of RAW264.7 cells into osteoclasts.