不同浓度牛乳铁蛋白对成骨细胞与破骨细胞共培养的影响

Effects of Different Concentrations of Bovine Lactoferrin on Osteoblast/Osteoclast Co-cultures

研究证实牛乳铁蛋白具有成骨活性功能,既能刺激成骨细胞增殖,也能诱导破骨细胞凋亡。骨骼的生长发育是一动态平衡的生物过程,是通过成骨细胞诱导的骨生成和破骨细胞诱导的骨吸收所调节的。采用成骨细胞MC3T3-E1细胞与破骨前体细胞RAW264.7细胞1∶1的比例直接接触的共培养方式,研究不同浓度(20、100、500μg/m L)的牛乳铁蛋白对共培养中成骨细胞和破骨细胞活性以及破骨细胞骨吸收功能的影响。首先,通过碱性磷酸酶(ALP)活性检测试剂盒检测共培养上清中的ALP活性,发现低浓度的牛乳铁蛋白能够抑制成骨细胞的ALP活性,而中高浓度的牛乳铁蛋白能够显著地促进成骨细胞的ALP活性。利用TRAP活性检测试剂盒及ELISA检测试剂盒,则发现低中浓度的牛乳铁蛋白能够促进破骨细胞的TRAP活性,而高浓度的牛乳铁蛋白能够显著地抑制破骨细胞的TRAP活性和共培养体系RANKL/OPG的比值。最后,通过骨吸收陷窝实验,证实了中高浓度组的牛乳铁蛋白能够显著地抑制破骨细胞的骨吸收功能。结果表明了高浓度的牛乳铁蛋白在共培养体系中不仅能够促进成骨细胞的活性,并且对破骨细胞具有抑制活性及功能的作用。

The osteogenic activity of bovine lactoferrin has been proved that bovine lactoferrin could promote proliferation and differentiation of osteoblasts and inhibit apoptosis of osteoclast. The growth and development of bones depended on the balance between bone resorption and bone formation. Osteoblasts and osteoclasts were associated with bone formation and bone resorption, respectively. The effects of different concentrations of bovine lactoferrin （20,100,500μg/mL） on the osteoblast and osteoclast activity and bone resorptive activity of osteoclast were studied by co-cultures of MC3T3-E1 cells and RAW264.7 cells （used at a ratio of 1：1） in this paper. Alkaline Phosphatase（ALP） activity was detected by ALP activity Assay Kit. The result showed that low concentrations of bovine lactoferrin inhibited ALP activity of osteoblasts, whereas middle and high concentra-tions of bovine lactoferrin could significantly promote ALP activity. By tartrate resistant acid phosphatase （TRAP） activity Assay Kit and the ELISA kits, it was found that low and middle concentration of bovine lacto-ferrin could promote the TRAP activity of osteoclasts, while high concentrations of bovine lactoferrin could sig nificantly inhibit the TRAP activity and the ratio of receptor activator of the NF-κB ligand （RANKL） and os-teoprotegerin（OPG）. Bone resorptive activity of osteoclast as evaluated on bone-mimetic plates was significant-ly inhibited by middle and high concentration of bovine lactoferrin. In general, these results indicated that high concentrations of bovine lactoferrin could not only promote osteoblasts activity, but also inhibit steoclasts activi-ty and function in co-culture system.