| The proliferation of osteoblasts plays an important role in bone health,especially in promoting the growth and development of infants.Studies have found that lactoferrin can effectively promote the proliferation of osteoblasts.Human milk is the most ideal source of lactoferrin for infants.However,for infants who can only drink milk substitute--infant formula,they can't get human lactoferrin.Therefore,it has great significance to study the physiological function of lactoferrin in promoting the proliferation of osteoblasts and its application in replacing human lactoferrin.In this study,Fe Cl2 and Fe Cl3 were used to synthesize magnetic nanoparticles(MNPs)by coprecipitation under alkaline conditions.Concanavalin A,lactoferrin specific adsorption ligand,was coated to the surface of magnetic nanoparticles by ultrasonic.Modified MNPs were characterized,the results showed that the seven characteristic peaks of MNPs were consistent with those of Fe3O4 by X-ray diffraction and the crystal lattice was confirmed to be Fe3O4,the morphology of the MNPs were regular and uniform in size confirmed by scanning electron microscopy and transmission electron microscopy.The average particles size of MNPs was 9.77±0.02 nm,and the magnetic intensity was 5.11 emu/g confirmed by laser particle size meter and magnetic intensity vibrometer.Skimmed milk or whey was used as raw material,the optimal process of recovery lactoferrin,adsorption specificity,target protein purity,recovery rate and storage method of MNPs were discussed.The results showed that optimal adsorption-elution time of lactoferrin were 4 min and 5 min,respectively,and the maximum adsorption capacity was 90 ?g/mg.The recovery rate of MNPs in both whey and eluent was higher than 99.99%,the recovery rate of lactoferrin was 92.13% with purity of 93.06%.The best storage way of MNPs was suspended in PBS at 4 ?.The extraction method had no effect on the iron saturation of lactoferrin and proliferation activity of osteoblasts.Bovine lactoferrin was used as the target,human lactoferrin was used as the control,the osteoblast MC3T3-E1 was used as the cell line.The proliferation activity of bovine lactoferrin was conducted through experiments including cell proliferation,inhibition of apoptosis,secretion of alkaline phosphatase,regulation of cell cycle,m RNA expression and translation of proliferating nuclear antigen(PCNA),and the express regulation of key proteins phosphorylation level in the MAPK pathway.The results showed that bovine lactoferrin can significantly promote osteoblast proliferation(P<0.05),suppress the apoptosis with the serum withdrawal(P<0.05),increase the secretion of alkaline phosphatase(P<0.05),shorten the cell cycle in G1/G0 phase,prolong the G2/M phase and S phase(P<0.05),improve the m RNA expression and translation of PCNA(P<0.05),up-regulate the phosphorylation of ERK1/2 and down-regulate of JNK and p-38 in the MAPK pathway to promotes the proliferation of osteoblast(P<0.05).The proliferation activity of bovine lactoferrin was significantly higher than that of human lactoferrin in all these aspects mentioned above(P<0.05).Bovine lactoferrin was modified with glycosylation to verify and screen the proliferation activity of glycosylation products that might be produced during pasteurization.MS/MS was used to measure the sites of glycosylation reaction.The two glycosylation products,lactoferrin reacted with fructose at a mass ratio of 1:1 for 24 h(24F1)and with galactose at a mass ratio of 1:1 for 48 h(48A1),had more significant proliferation effect than that of unmodified lactoferrin(P<0.05).The common binding sites of 24F1 and 48A1 were mainly occur in lysine at number 47 th,329th and 627 th,while 24F1 has unique binding sites in lysine at number 288 th and 471 th,and 48A1 in lysine at number 581 th.MAPK pathways of 24F1 and 48A1 promoting the proliferation of osteoblasts were consistent with those of bovine lactoferrin and human lactoferrin,respectively. |
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