番茄红素对活性氧族介导破骨细胞骨吸收功能的影响

Effect of lycopene on reactive oxygen species-mediated bone resorption by osteoclasts

背景:研究表明,番茄红素能够促进成骨细胞的增殖和生长,增加其矿化能力。目的:实验拟验证抗氧化剂番茄红素影响活性氧族介导破骨细胞骨吸收功能的作用途径。设计、时间及地点:随机分组设计,对比观察,实验于2005-01/2006-12在山东省骨科研究所完成。材料:24h内出生的清洁级Wistar大鼠幼鼠30只,用于破骨细胞的培养;番茄红素由Sigma公司提供。方法:将24孔细胞培养板内细胞分为5组。空白对照组:所用培养液不含番茄红素;10-7mol/L番茄红素组、10-6mol/L番茄红素组、10-5mol/L番茄红素组和10-5mol/L维生素C组培养孔内预先置入盖玻片或薄骨片,对种植其上的破骨细胞分别用含有不同浓度番茄红素或维生素C的α-MEM培养基进行培养。主要观察指标:在分离培养的细胞玻璃爬片或骨片进行抗酒石酸酸性磷酸酶染色、四唑氮蓝染色,最后对骨片进行扫描电镜照像并用image-proplus5.0图像分析软件分析骨吸收陷窝的数目和面积。结果:①抗酒石酸酸性磷酸酶染色阳性多核细胞镜下胞浆酸性磷酸酶活性部位呈紫红色,细胞核染色阴性或淡染,伪足清晰。②四唑氮蓝染色结果提示,10-5mol/L的番茄红素明显抑制了破骨细胞产生活性氧族的能力。③扫描电镜结果显示,破骨细胞在骨片上形成形态不一的骨陷凹,10-7mol/L的番茄红素部分抑制了骨吸收陷凹面积的增加,而10-5mol/L的番茄红素则几乎完全抑制了骨吸收陷凹的形成。结论:番茄红素在体外可以通过抑制破骨细胞产生活性氧族来抑制其骨吸收功能。

BACKGROUND： Some studies show that lycopene can promote osteoblast proliferation and growth and enhance osteoblastic mineralization. OBJECTIVE： To explore the action pathway of lycopene, a kind of antioxidant, on reactive oxygen species （ROS）-mediated bone resorption by osteoclast. DESIGN, TIME AND SETTING： Randomized comparative observation was performed at Shandong Institute of Orthopedics from January 2005 to December 2006. MATERIALS： Thirty clean-grad neonate Wistar rats born within 24 hours were selected for culture of osteoclasts; lycopene was provided by Sigma, USA. METHODS： Osteoclasts isolated from the long bones of neonatal Wistar rats were planted into 24-well plates and randomly divided into 5 groups： blank control group with no lycopene; 10^- 7 mol/L, 10^-6 mol/L and 10^-5 mol/L lycopene groups, and 10^-5 mol/L vitamin C group were cultured in the culture plate （coverslip or bone flaps had been placed in the wells already） and a -MEM supplemented with varying doses of lycopene or vitamin C. MAIN OUTCOME MEASURES： The coverslips or bone flaps with cells were stained with tartrate-resistant acid phosphatase （TRAP） and nitroblue tetrazolium （NBT）. The number and area of resorption lacuna were analyzed by the image-pro plus5.0 image analyzing software. RESULTS： In the TRAP positive multinucleated cells, the active area with TRAP was stained mauve, while the cell nucleuses stained negatively or stained lightly. The parapodium of osteoclasts was distinct. The NBT staining showed that lycopene （10^-5 mol/L） significantly inhibited the production of ROS in osteoclasts. The bone flaps were absorbed by osteoclasts and formed lots of lacuna with different appearances under the scanning electron microscope. Lycopene at a concentration of 10.7 mol/L partially reduced the area of resorption lacuna, whereas 10^-5 mol/L lycopene almost completely inhibited the resorption function. CONCLUSION： Lycopene can inhibit the bone resorption of osteoclasts in vitro by decreasing the production of reactive oxygen species.