摘要：

目的：探究低氧状态下低氧诱导因子-1α（hypoxia inducible factor-1α, HIF-1α）对骨细胞凋亡的影响。方法：用氯化钴（cobalt dichloride, CoCl₂）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CoCl₂对MLO-Y4细胞增殖活性的影响；Hoechst 33 342及TUNEL染色检测CoCl₂处理后的MLO-Y4细胞的凋亡情况；利用实时荧光定量聚合酶链反应（RT-qPCR）、蛋白质印迹（Western blot）和细胞免疫荧光检测CoCl₂作用下MLO-Y4细胞表达HIF-1α与凋亡相关因子Caspase-3及Bcl-2的情况；检测HIF-1α siRNA转染对MLO-Y4细胞表达HIF-1α和促凋亡相关因子Caspase-3的影响。结果：200 μmol/L CoCl₂作用24 h后，MLO-Y4细胞的增殖率显著下降，并呈浓度和时间依赖性。200 μmol/L CoCl₂作用24 h可上调MLO-Y4细胞HIF-1α的表达，并促进MLO-Y4骨细胞凋亡；siHIF-1α转染后，MLO-Y4细胞HIF-1α表达下降，促凋亡基因Caspase-3也表现为相同的下降趋势，骨细胞凋亡减少。结论：HIF-1α在体外化学模拟低氧条件下，可促进骨细胞凋亡。

关键词: 氯化钴, 骨细胞, HIF-1α, 细胞凋亡

Abstract:

Objective: To investigate the effects of hypoxia inducible factor-1α(HIF-1α) on osteocytes apoptosis under the hypoxic state. Methods: The osteocyte-like cell line MLO-Y4 cells were stimulated by cobalt dichloride(CoCl₂) to establish a hypoxic culture system in vitro. CCK-8 assays were used to detect the effect of CoCl₂ on the proliferation activity of MLO-Y4 cells. Hoechst 33 342 and TUNEL staining were used to confirm the apoptosis of MLO-Y4 cells treated with CoCl₂. RT-qPCR, Western blot and immunofluorescence techniques were used to detect the expression of HIF-1α and apoptosis related factors (Caspase-3 and Bcl-2) in MLO-Y4 cells. The effect of HIF-1α siRNA transfection was based on the expression of HIF-1α and pro-apoptosis factor Caspase-3 by MLO-Y4 cells. Results: The proliferation rate of MLO-Y4 cells was significantly decreased in MLO-Y4 cells treated by 200 μmol/L CoCl₂ for 24 h, and showed in a concentration and time dependent manner. CoCl₂ (200 μmol/L) could up-regulate the expression of HIF-1α in MLO-Y4 cells and promote the apoptosis process. After siHIF-1α transfection, the expression of HIF-1α and proapoptotic gene Caspase-3 were decreased, as well as the apoptosis of osteocytes. Conclusion: HIF-1α promotes the apoptosis of osteocyte-like cell line MLO-Y4 cells under chemical hypoxia in vitro.

Key words: cobalt dichloride, osteocytes, hypoxia inducible factor-1α, cell apoptosis