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

摘要

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 21days, 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 100μg/mL concentration of LF.2) The results of ALP staining showed that the highest ALP activity was achieved on the 21st 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.%目的：探索乳铁蛋白促进大鼠成骨细胞增殖的最佳浓度及1000μg／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1000μg／mL，在1、3、5、7d后分别采用CCK－8法测定细胞增殖；用1000μg／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，1000μg／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；1000μg／mL的乳铁蛋白促进成骨细胞分化作用呈时间依赖性。