抗坏血酸抑制核因子κB受体活化子配体诱导RAW264.7细胞的分化和功能  

Ascorbic acid inhibits the formation and function of osteoclasts from RAW264.7 cells induced by receptor activated nuclear factor kappaB ligand in vitro

作　　者：肖新华[1] 周厚德[1] 袁凌青[1] 谢辉[1] 廖二元[1] 

机构地区：[1]中南大学湘雅二医院代谢内分泌研究所,长沙410011

出　　处：《中华医学杂志》2004年第24期2102-2106,共5页National Medical Journal of China

基　　金：国家自然科学基金资助项目(39970347)

摘　　要：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 observe the influence of ascorbic acid (AA) on osteoclastogenesis induced by receptor activated nuclear factor kappaB ligand (RANKL) in RAW264.7 cells and to study the mechanism of AA in osteoclast differentiation Methods Mouse mononuclear cells of the line RAW264 7 were cultured AA and/or RANKL were added Cellular respiration of RAW264.7 cells was assayed by MTT cell proliferation method. RAW264 7 cells were inoculated and RANKL and AA of different concentrations were added TRAP staining was used to observe the formation of osteoclasts RAW264 7 cells were inoculated on OAAS, RANKL and AA were added The number of bone absorption pits was counted RT PCR was used to measure the expression of RANK, matrix metalloproteinase 9, TRAF6, TRAP, integrin αv, integrin β3 , cathepsin K, and carbonic anhydrase Ⅱ (CA Ⅱ) Results AA of the concentration of 500μg/ml significantly inhibited the proliferation of RAW264 7 cells (0 13±0 05 vs 1 11±0 22, P <0 05) RANKL significantly inhibited the proliferation of RAW264 7 cells as well (1 58±0 22 vs 1 26±0 17) AA showed no synergistic action to RANKL AA alone could not induce the formation of TRAP positive osteoclasts from RAW264 7 cells and 0～100 μg/ml AA inhibited the RANKL induced formation of osteoclast like multinuclear cells from RAW264 7 cells AA alone up regulated the mRNA expression of CA Ⅱ, cathepsin K, and TRAP by 10, 1 5, and 1 5 times respectively RANKL plus AA significantly down regulated the mRNA expression of CAII and RANK by 60% and 20% respectively No bone absorption pit was seen after addition of AA The number of pits on OAAS after addition of AA+RANKL was significantly lower than that after addition of RANKL alone ( P <0 05) Western blotting showed that no difference was seen in the protein expression of CAⅡ between the RANKL group and the control group, however, the protein expression of CAⅡ in the low concentration AA+RANKL group was significantly lower than that in the RANKL alone group ( P <0 05) Conclusion AA directly inhibits

关 键 词：RAW264.7 破骨细胞 RANKL 诱导 核因子KB 抗坏血酸 抑制 受体活化 分化 功能基因 

分 类 号：R33[医药卫生—人体生理学]