目的比较研究柚皮苷及其代谢物柚皮素对体外培养乳鼠颅骨成骨细胞分化成熟的影响。方法无菌条件下剥离新生SD大鼠颅骨，采用Ⅱ型胶原酶多次消化，分离得到原代乳鼠颅骨成骨细胞，待原代细胞铺满80%皿底时行传代培养。噻唑蓝法检测细胞的增殖情况，碱性磷酸酶活性的高低作为检测药物影响乳鼠颅骨成骨细胞分化成熟的指标，筛选得到最佳的药物浓度。在乳鼠颅骨成骨细胞的培养体系中添加最佳浓度的柚皮苷和柚皮素，酶联免疫分析法检测柚皮苷组、柚皮素组和对照组之间骨钙素、骨形态发生蛋白-2、骨桥蛋白和I型胶原的分泌量，比较钙化结节数量的差异，并用实时定量荧光逆转录聚合酶链反应法检测成纤维细胞生长因子、胰岛素样生长因子-1、Runx-2、Osterix、OPG及RANKL的基因表达情况。结果柚皮苷和柚皮素均能明显提高乳鼠颅骨成骨细胞的碱性磷酸酶活性，刺激骨钙素、骨形态发生蛋白-2、骨桥蛋白和I型胶原的分泌，增加钙化结节数量，并促进成骨相关基因的mRNA表达，但柚皮素的活性明显高于柚皮苷。结论柚皮素促进乳鼠颅骨成骨细胞分分化成熟的活性显著高于柚皮苷，说明柚皮苷经代谢后，其代谢产物柚皮素的促骨形成活性得到增强，在抗骨质疏松新药的开发及剂型设计方面均具有一定的指导意义。
Abstract
OBJECTIVE To investigate the effects on the differentiation and maturation of rat osteoblast(ROB) by naringin and it′s metabolite-naringenin. METHODS Primary ROB were obtained from new born SD rat skull after dissected and digested many times by type II collagenase during sterile condition. Serial subcultivation were proceeded when cells covered 80% culture dish. Cell proliferation was detected by MTT,while the alkaline phosphatase (ALP) activity was adopted as osteogenic differentiation marker to screen the best concentration of naringin and naringenin. The secretion of osteocalcin,bone morphogenetic protein-2(BMP-2),oesteopontin(OPN) and collagen I,the bone mineralized nodulus,even the gene expression of bFGF,IGF-1,Runx-2,Osterix,OPG and RANKL all were compared among the naringin-supplemented group,naringenin-supplemented group and the control. RESULTS Both naringin and naringenin can significantly improved ALP activity,the secretion of osteocalcin,BMP-2,OPN and collagen I,the bone mineralized nodulus also were raised. Besides,these two drugs also stimulated the expression of genes which related to the osteogenesis of ROB. However,naringenin is stronger than naringin in above markers significantly. CONCLUSION The osteoprotective effects of naringenin is stronger than naringin at enhancing the osteogenic differentiation of ROB,suggesting that naringin can be administered via oral and its metabolites developed higher activity to prevent osteoporosis. These results may provide a guide for the new drug develop and dosage forms design during osteoporosis therapy.
关键词
柚皮苷 /
柚皮素 /
成骨细胞 /
分化 /
成熟
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Key words
naringin /
naringenin /
rat osteoblast /
differentiation /
maturation
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中图分类号：
R915
R965
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参考文献
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脚注
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基金
国家科技重大专项重大新药创制(2011ZX09401-308-28)；国家自然科学基金资助项目(81073037)；国家自然科学基金资助项目(31071043)；陕西省中医管理局中医药科研课题(2011ZY44)
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