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目的研究抗坏血酸对核因子κB受体活化子配体(RANKL)诱导体外培养的破骨前体细胞RAW2647形成成熟多核破骨细胞的影响,探讨抗坏血酸在破骨前体细胞分化中的作用及机制。方法利用不同浓度的抗坏血酸与RANKL单独或共同处理RAW2647细胞,MTT法测定细胞增殖,抗酒石酸酸性磷酸酶(TRAP)染色法观察TRAP阳性多核细胞,逆转录聚合酶链反应(RTPCR)测定破骨细胞表型基因和功能基因的表达,Western印迹方法检测碳酐酶Ⅱ基因蛋白表达,破骨细胞的骨吸收功能用骨吸收陷窝面积计数法分析。结果抗坏血酸和RANKL都抑制RAW2647细胞的增殖(P<005),但它们之间无协同作用。抗坏血酸本身不能诱导RAW2647细胞形成破骨细胞,但可抑制RANKL诱导的TRAP阳性多核破骨细胞形成(P<005)。抗坏血酸下调RANKL诱导的碳酐酶Ⅱ和RANK基因mRNA表达及碳酐酶Ⅱ基因蛋白表达,抑制骨吸收功能(P<005)。结论抗坏血酸能直接抑制RANKL诱导的破骨细胞形成和功能。
Objective To investigate the effect of ascorbic acid on the formation of mature multinucleated osteoclasts induced by RANKL induced osteoclast precursor RAW2647 in vitro and to explore the role and mechanism of ascorbic acid in differentiation of osteoclast precursors. Methods RAW2647 cells were treated with different concentrations of ascorbic acid and RANKL alone or in combination with each other. MTT assay was used to detect the cell proliferation. TRAP positive cells were detected by TRAP staining and RTPCR was used to detect osteoclasts Phenotypic and functional gene expression, Western blotting was used to detect the expression of carbonic anhydrase Ⅱ gene protein, and osteoclast bone resorption function was analyzed by counting bone resorption area. Results Both ascorbic acid and RANKL inhibited the proliferation of RAW2647 cells (P <005), but there was no synergistic effect between them. Ascorbate itself failed to induce RAW2647 cells to form osteoclasts, but inhibited RANKL-induced TRAP-positive multinucleated osteoclasts (P <005). Ascorbic acid down-regulated RANKL-induced mRNA expression of carbonic anhydrase II and RANK, and expression of carbonic anhydrase II gene, and inhibited bone resorption (P <005). Conclusion Ascorbic acid can directly inhibit RANKL-induced osteoclast formation and function.