乳铁蛋白促进成骨细胞增殖的最佳浓度及促进分化的最佳时间

The best concentration and the best time of lactoferrin for the stimulation of osteoblast proliferation and differentiation

目的探索乳铁蛋白促进大鼠成骨细胞增殖的最佳浓度及1 000μg/mL乳铁蛋白对促进成骨细胞分化的最佳时间。方法用混合酶消化法分离大鼠颅盖骨成骨细胞进行原代培养;不同浓度乳铁蛋白干预成骨细胞,浓度分别为0、0.1、1、10、100、200、400、600、800和1 000μg/mL,在1、3、5、7d后分别采用CCK-8法测定细胞增殖;用1 000μg/mL的乳铁蛋白干预成骨细胞7、14、21 d后,采用碱性磷酸酶染色法检测细胞内碱性磷酸酶的活性。结果 1.CCK-8法测定细胞增殖结果显示,低于100μg/mL浓度乳铁蛋白组其OD值随着浓度的增高而增高,而高于100μg/mL浓度乳铁蛋白组其OD值随着乳铁蛋白浓度的增高而递减;2.碱性磷酸酶染色法结果显示,1 000μg/mL的乳铁蛋白在干预第21天时碱性磷酸酶活性最高。结论 100μg/mL乳铁蛋白是促进大鼠成骨细胞增殖的最佳浓度;1 000μg/mL的乳铁蛋白促进成骨细胞分化作用呈时间依赖性。

Objective To explore the optimal concentration of the lactoferrin（LF） for the stimulation of rat osteoblast proliferation and the best time to promote osteoblast differentiation using 1000μg/ml lactoferrin.Methods Primary rat osteoblasts were harvested with mixed enzyme from neonatal calvaria of Sprague Dawley rats and then cultured.After the treatment with different concentrations of LF（0,0.1,1,10,100,200,400,600,800,and 1 000 μg/mL） for 1,3,5,and 7 days,the cell proliferation was analyzed using CCK-8 methods.After the treatment with LF of 1000μg/mL for 7,14,and 21 days,alkaline phosphatase staining method was used to evaluate the ALP activity of the osteoblast.Results 1） The results of cell proliferation with CCK-8 method showed that below 100 μg/mL concentration of LF,OD value increased as the concentration increased.However,OD value decreased with the concentration of LF increased using over 100 μg/mL concentration of LF.2） The results of ALP staining showed that the highest ALP activity was achieved on the 21 st day using 1 000 μg/mL concentration of LF.Conclusion The best concentration of LF for rat osteoblast proliferation is 100 μg/mL.LF of 1 000 μg/mL promotes osteoblast differentiation in a time dependent manner.