缺氧诱导因子-2α调控破骨细胞分化及功能的研究

Regulation of hypoxia-inducible factor-2αon osteoclast differentiation and function

目的:观察缺氧环境对破骨细胞形成的影响,探讨缺氧诱导因子-2α(HIF-2α)在其中的调控作用。方法:在正常氧浓度、5%O 2浓度和1%O 2浓度条件下诱导培养破骨细胞,抗酒石酸酸性磷酸酶(TRAP)染色后进行计数。Western blot检测缺氧条件下破骨细胞中HIF-2α的表达。应用HIF-2α抑制剂PT2385(5μmol/L)抑制HIF-2α活性后,实时荧光定量聚合酶链反应(RT-qPCR)检测破骨细胞功能相关基因的表达。结果:TRAP染色显示骨髓基质单核细胞(BMM)可成功诱导成多核的破骨细胞;破骨细胞的数目在正常氧组为(55.1±8.2)个,5%O 2低氧组为(72.5±10.7)个,1%O 2低氧组为(94.7±15.6)个,差异有统计学意义(F=8.341,P<0.05)。破骨细胞在低氧条件下HIF-2α的蛋白表达增高,5%O 2低氧组为正常氧组的3.1倍,1%O 2低氧组为正常氧组的6.5倍,差异有统计学意义(F=21.070,P<0.05)。TRAP、组织蛋白酶K(CK)、基质金属蛋白酶-9(MMP-9)、活化T细胞核因子1(NFATc1)基因表达在5%O 2低氧组升高,PT2385(5μmol/L)可抵消低氧这种作用,差异有统计学意义(P<0.05)。结论:缺氧环境可以通过激活HIF-2α促进破骨细胞的形成和分化,阻断HIF-2α可抑制破骨细胞的骨吸收功能。

Objective To observe the effect of hypoxia on osteoclast formation,and to explore the regulatory role of hypoxia inducible factor-2α(HIF-2α).Methods Osteoclasts were cultured in normal oxygen concentration,5%O2 concentration and 1%O2 concentration,and counted after tartrate-resistant acid phosphatase(TRAP)staining.The expression of HIF-2αin osteoclasts under hypoxia condition was detected by Western blotting.After HIF-2αactivity was inhibited by HIF-2αinhibitor PT2385(5μmol/L),the expression of genes related to osteoclast function was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Results TRAP staining showed that bone marrow mononuclear cells(BMM)could induce osteoclasts successfully.The number of osteoclasts in the normal oxygen group was(55.1±8.2),(72.5±10.7)in the 5%O2 hypoxia group,and(94.7±15.6)in the 1%O2 hypoxia group,with the difference being statistically significant(F=8.341,P<0.05).The protein expression of HIF-2αin osteoclasts was increased under hypoxic conditions.The expression of HIF-2αin 5%O2 hypoxic group was 3.1 times higher than that in the normal oxygen group,and that in the 1%O2 hypoxic group was 6.5 times higher than that in the normal oxygen group,with statistically significant difference(F=21.070,P<0.05).The expression of TRAP,cathepsin K(CK),matrix metalloproteinase-9(MMP-9),and activated T nuclear factor 1(NFATC1)genes increased in the 5%O2 hypoxia group,and PT2385(5μmol/L)could counteract the effect of hypoxia,and the difference was statistically significant(P<0.05).Conclusion Hypoxic environment can promote the formation and differentiation of osteoclasts by activating HIF-2α,and blocking HIF-2αcan inhibit the bone resorption of osteoclasts,suggesting that HIF2-αmay be a therapeutic target for osteoporosis.