柚皮苷对体外培养乳鼠颅骨成骨细胞增殖和分化成熟的影响

Effects of naringin on proliferation,differentiation and maturation of rat calvarial osteoblastsin vitro

目的:研究柚皮苷对体外培养大鼠乳鼠颅骨成骨细胞(rat calvarial osteoblasts,ROB)增殖和分化成熟的影响。方法:取新生SD大鼠颅骨,多次酶消化法培养ROB,每3 d换液1次,铺满80%皿底后传代培养。采用终浓度分别为1×10-4,1×10-5,1×10-6,1×10-7mol·L-1的柚皮苷进行药物干预,MTT法检测细胞增殖率,同时检测成骨性诱导培养9 d后的碱性磷酸酶(alkaline phosphatase,ALP)活性,筛选出最佳浓度。以最佳药物浓度干预ROB的分化成熟,比较柚皮苷组和不加药的对照组之间的骨钙素(osteocalcin,OC)、骨桥蛋白(oesteopontin,OPN)、骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)的分泌量;提取总RNA,Real-time RT-PCR检测bFGF,IGF-1,Runx-2,Osterix,ERα和ERβ的mRNA水平;Western-blot法检测ERα,ERβ和Ⅰ型胶原蛋白的表达量;并用雌激素竞争性抑制剂ICI 182.780进行阻断,观察阻断前后各指标的变化情况。结果:柚皮苷剂量依赖性刺激成骨细胞的增殖,并可强烈促进ROB的分化成熟。最佳药物浓度为1×10-5mol·L-1,该浓度下的柚皮苷可明显提高OC,OPN和BMP-2的分泌量,促进与成骨相关的因子bFGF,IGF-1,Runx-2,Osterix和雌激素受体基因ERα及ERβmRNA的表达,同时也可增强ERα,ERβ和Ⅰ型胶原蛋白的分泌,但以上效果均可被阻断剂ICI 182.780所抑制。结论:浓度为1×10-5mol·L-1的柚皮苷可显著促进ROB的增殖与分化成熟,并且该促进效果会被雌激素通路阻断剂ICI 182.780所抑制,表明柚皮苷属于植物雌激素,通过雌激素信号通路发挥其促骨形成活性,可以用于骨质疏松症的防治。

Objective： To investigate the effects of naringin on the proliferation, differention and maturaion of rat calvarial os- teoblasts （ROB）. Method： Segregated neonatal SD rat skull, enzyme digestion to obtain ROB. The culture medium was replaced ev- ery three days. Serial subcultivation proceeded when cells covered with 80% culture dish. Naringin supplemented into the culture at 1 ×10^-4 ,1 ×10^-5 ,1 ×10^-6,1 ×10^-7 mol · L^-1 respectively. MTT method was adopted in proliferation analysis and the activity of ALP was examined after induced 9 days. Search the best concentration and supplemented into the medium, then the osteogenic differ- entiation markers including the secretion amount of osteocalcin, osteopontin and bone morphogenetic protein-2 were compared between the naringin-supplemented group and the control. Total RNA was isolated and the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERa and ERfl was investigated by Real time RT-PCR. Total protein also was isolated and the expression ERa, ERfl and collagen I was examined by Western blot. After the addition of ICI 182. 780, an inhibitor of the estrogen signal pathway, these index also was exam- ined and the changes were compared. Result： The ROB proliferation was motivated by naringin dose-dependently. And it evidently leads to osteogenic process and maturation. 1 ×10^-5 mol· L^-1 is the best concentration. Naringin improved the secretion of osteocal- ein, osteopontin, bone morphogenetic protein-2 and collagen l significantly. Besides, it can also enhanced the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERa and ERfl. While all these effects can be restrained by ICI 182. 780. Conclusion： The naringin with fi- nal concentration of 1 ×10^-5 mol · L^- 1 enhances the osteogenic differentiation and maturation of ROB significantly, while the promoting effects vanished after the addition of ICI 182. 780. These results suggesting that naringin is one of the phytoestrogens and have the ac- tivity of bone formation may via estrogen signal pathway, it can be developed into a new drug for osteoporosis therapy.