Abstract:

Objective: To investigate the effect of hypoxia-inducible factor-1α (HIF-1α) on the changes of osteoclast bone resorption capacity by regulating Rac1 on cytoskeleton under hypoxic environment. Methods: The hypoxia culture system was established by stimulating mouse mononuclear cell line RAW264.7 with cobalt dichloride (CoCl₂) in vitro. The differentiation of RAW264.7 cells was induced by sRANKL conditioned culture medium and stimulated by CoCl₂ simulated hypoxic environment, and TRAP staining was performed after 7 days; real-time quantitative polymerase chain reaction (RT-qPCR), cellular immunofluorescence assay and Western blotting were used to detect the expression of HIF-1α KO Rac1, osteoclast-related surface markers and closure zones in RAW264.7 under hypoxia. Simultaneously, HIF-1α conditional knockout cell line RAW264.7 and the effect of the inhibitor of Rac1 on the expression of osteoclast-related genes and closure zones in RAW264.7 under the same conditions were detected.Results: Hypoxia could increase the volume of RAW264.7 induced osteoclasts and enhance bone resorption capacity (P<0.01). The level of Rac1 mRNA expressed by osteoclasts in hypoxia group was increased, and the expression of closure zones were significant (P<0.001). The volume of osteoclasts induced by HIF-1α KO RAW264.7 cells decreased, the expression of HIF-1α was significantly decreased, and Rac1 was also decreased (P<0.001); meanwhile, the volume of osteoclasts decreased in the inhibitor group, the expression of HIF-1α remained unchanged, and the closure zones formed abnormally (P<0.01). Conclusion: Under hypoxia, mouse mononuclear cell line RAW264.7 can regulate Rac1 through HIF-1α protein to affect the cytoskeleton of osteoclasts, and then change the bone resorption capacity of osteoclasts.

Key words: cytoskeleton, hypoxia-inducible factor-1α, RAW264.7 cells, Rac1

摘要：

目的：探究低氧条件下低氧诱导因子-1α（hypoxia-inducible factor-1α，HIF-1α）通过调控Rac1对细胞骨架的影响及其调节破骨细胞骨吸收功能变化的作用。方法：利用氯化钴（cobalt dichloride，CoCl₂）刺激小鼠单核细胞系RAW264.7建立体外低氧破骨细胞培养体系。采用sRANKL条件培养液诱导RAW264.7细胞分化，利用CoCl₂模拟低氧环境刺激细胞，7 d后行TRAP染色；利用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）、细胞免疫荧光检测和蛋白质印迹法（Western blotting）检测低氧下破骨细胞表达HIF-1α、Rac1及破骨细胞相关表面标志物与封闭带的情况；同时检测HIF-1α条件性敲除细胞株RAW264.7及Rac1活性抑制剂对相同条件下破骨细胞相关基因和封闭带的影响。结果：低氧环境可促使RAW264.7诱导形成的破骨细胞体积增大、骨吸收能力增强（P<0.01）。低氧组破骨细胞表达的Rac1 mRNA水平升高，且封闭带表达显著（P<0.001）。HIF-1α KO RAW264.7细胞诱导形成破骨细胞体积减小，HIF-1α表达显著降低，Rac1也随之降低（P<0.001）；同时，抑制剂组诱导生成的破骨细胞体积减小，HIF-1α表达保持不变，封闭带形成异常（P<0.01）。结论：小鼠单核细胞系RAW264.7在低氧环境下可通过HIF-1α调控Rac1来影响其所形成的破骨细胞细胞骨架，进而改变破骨细胞的骨吸收能力。

关键词: 细胞骨架, 低氧诱导因子-1α, RAW264.7细胞, Rac1

CLC Number: 

• R782