分子氢对RANKL和RANKL/TNF-α诱导的RAW264.7细胞向破骨细胞分化的影响

摘要

目的 探讨分子氢(Hydrogen,H2)体外对RANKL和RANKL/TNF-α诱导小鼠单核细胞RAW264.7向破骨细胞分化的影响.方法 体外培养小鼠单核细胞RAW264.7,在RANKL和RANKL/TNF-α的作用下诱导分化为破骨细胞,并用分子氢进行干预,用CCK-8法测定H2对RAW264.7细胞增生活性的影响,检测各组经诱导后所得破骨细胞的氧化应激状态,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法观察TRAP阳性破骨细胞的形态并计数,实时定量PCR法检测细胞转录因子组织蛋白酶K(cathepsin K, CTK)和金属蛋白酶-9(matrix metalloprotein-9, MMP-9)mRNA的表达.结果 含分子氢培养基不影响RAW264.7细胞的增殖活性,降低破骨细胞的氧化应激状态;RANKL和RANKL/TNF-α诱导的RAW264.7细胞TRAP染色阳性细胞数,经分子氢干预作用下明显降低(P<0.05);含分子氢培养基能抑制RANKL和RANKL/TNF-α诱导小鼠单核细胞RAW264.7胞内CTK和MMP-9的表达(P<0.05).结论 分子氢能降低破骨前体细胞的氧化应激状态,从而抑制向破骨细胞分化.%Objective To discuss the effect of H2 on osteoclast differentiation of RAW264.7 cells induced by RANKL and RANKL/TNF-α.MethodsRAW264.7 cells were treated with H2 in the presence of RANKL and RANKL/TNF-α.RAW264.7 cells viability was assessed by CCK-8.Test the Oxidative Stress of the induced RAW264.7.The number of TRAP-positive cells were counted under light microscopy.The levels of cathepsin K (CTK) and matrix metalloprotein-9(MMP-9) mRNA were analyzed by real-time PCR.ResultsH2 can not influence the RAW264.7 cell viability but can lower oxidative stress.The significant difference(P<0.05) indicated that H2 could significantly decrease the number of TRAP-positive MNCs.The significant difference among the 4 groups in CTK and MMP-9 genes (P<0.05) indicated that H2 could down-regulate their mRNA expression.ConclusionH2 can reduce the oxidative stress and inhibit differentiation of RAW264.7 cells into osteoclasts.