Effects Of Compositions Of Lactoferrin On Proliferation Activity Of Osteoblast

Lactoferrin is a bioactive protein which has been widespread used. Recently, it has been demonstrated to be an anabolic factor for bone. The effect of Lactoferrin on bone growth and metabolism can be applied to anti-osteoporosis and to reduce the relative complications. The biological functions of proteins depend on its structure. This study focuses on how lactoferrin molecule compositions affect the proliferation of osteoblast cells, for instance, the iron saturation level, the degree of glycosylation and the lactoferrin fragments and hydrolysates.In this study, the iron-binding ions of native bovine lactoferrin were removed or bound by dialysis. Deglycosylation was performed using Peptide N-glycosidase F, Lactoferrin fragments and hydrolysates were prepared by pepsin, trypsin and alkaline protease. We obtained the following samples: Apo-LF, Holo-LF, Degly-LF and ten different types of lactoferrin peptide. We determined the best interval of changing the water at seven times and 8h an interval for preparing the Apo-LF by ICP-AES. The best combination of iron dialysis interval is four times of water changes at the same interval of 8h. Monitored by SDS-PAGE, we can get the utmost split reaction of glycosylation. Combining SDS-PAGE and Tricine SDS-PAGE, we found that lactoferrin has a quite strong quality in anti-trypsin hydrolysis.Osteoblast cells were extracted from calvaria in newborn SD rats by enzyme digestion, the proliferation ration of osteoblast cells was determined by MTT assay. We observed that the stimulating osteoblast proliferation activity of LF in vitro decreased with increasing iron saturation level at 20 and 100μg/m L, as the culture growth over time, this advantage tend to expand. The osteoblast proliferation of Apo-LF gradually get inferior than Native-LF. Iron ions inhibit osteoblast cells growth. We also observed how lactoferrin of different iron saturation level access into the osteoblasts by laser scanning confocal microscope, the results show that there is no significant difference in the iron saturation level of lactoferrin. Degly- LF can not stimulate osteoblast proliferation activity as well as Native- LF, whose high concentrations at various points in time was significantly weaker than the natural state of lactoferrin, a low concentration of deglycosylation Lactoferrin at 24 h, 48 h not significantly contribute to bone cell proliferation, the concentration of deglycosylated lactoferrin only during 72 h can significantly promote the proliferation of osteoblasts, and the sugar chain alone time, did not significantly promote osteoblast proliferative activity. The LF fragment we got after three kinds protease hydrolysis 1h, 2h, 4h, osteogenic cell proliferation activity is weaker than the natural state of lactoferrin.This study describes the different proliferation of osteoblast cells of different structures of lactoferrin. This article laid theoretical basis on further study of structure-activity relationship of lactoferrin.