Different Concentration Of Lactoferrin Influence On The Generation Of Rat Osteoblast Proliferation

Objective:The objective of this study was to evaluate the effect of lactoferrin (LF) on primaryrat osteoblast proliferation, to explore the relationship of low concentration ofLactoferrin (<100ug/ml) and high concentration of Lactoferrin (>100ug/ml) andosteoblast proliferation, provide reference for the further study of Lactoferrin.Methods:(1) Primary rat osteoblasts were isolated by mixed enzyme which contains trypsin andcollagenaseⅠfrom neonatal rat calvarial bone，through the adoption of "differentialadherent method" for purification of osteoblast. Then observe the cell morphologyand growth condition, and identified using alkaline phosphatase dyeing and collagenⅠimmunohistochemical staining.(2) the second generation of osteoblast were added to96-well plates, and each holdhas3000cells. With the intervention of different concentrations of lactoferrin onosteoblasts, the experimental groups was divided into the following:0ug/ml,0.1ug/ml,1ug/ml,10ug/ml,100ug/ml,200ug/ml,400ug/ml,600ug/ml,800ug/ml,1000ug/ml. The cell proliferation are assayed by CCK-8method after1,3,5,7day.(3) Statistical methods: Experimental data using mean±standard deviation to show,SPSS17.0statistical software is applied for statistical analysis.The groups werecompared using one-way analysis of variance (ANOVA),there was statisticallysignificant when P﹤0.05.Results:(1) The cultured cells have the typical features of osteoblast，osteoblast spread outfully after24h with morphological diversity like, triangular, polygonal, long fusiform,have different length of protuberant, visible nuclei, and stretched a bulkypseudopodium; Alkaline phosphatase dyeing and collagenⅠimmunohistochemicalstaining are both positive.(picture) (2) The result of CCK8method shows that the medication groups of1ug/ml,10ug/ml,100ug/ml’OD value were significantly higher than the control group (p <0.01),200ug/ml,400ug/ml,600ug/ml group are lower than the control group (p <0.05),800ug/ml and1000ug/ml group are lower than the control group (p <0.01)after1day.0.1ug/ml,1ug/ml group OD value is higher than the control group, withstatistically significant (p <0.05), while10ug/ml,100ug/ml group were higher thanthe control group, with a significant statistical significance (p <0.01),200ug/mlgroup’s OD value is lower than the control group, but there was no statisticallysignificant (p>0.05),400ug/ml group is lower than the control group (p <0.05)，600ug/ml,800ug/ml and1000ug/ml groups are lower than the control group (p <0.01) after3day.0.1ug/ml,1ug/ml,10ug/ml group were higher than control group(p <0.05),100ug/ml group is significantly higher than control group (p <0.01),200ug/ml,400ug/ml,600ug/ml,800ug/ml and1000ug/ml significantly lower than thecontrol group (p <0.01) after5day.1ug/ml group was significantly higher thancontrol group (p <0.01), and0.1ug/ml,10ug/ml,100ug/ml group is higher than thecontrol group, with no statistical significance,200ug/ml,400ug/ml,600ug/ml,800ug/ml and1000ug/ml, significantly lower than the control group (p <0.01) after7day. At the same time, OD value was risen along with the increasing of theconcentration and processing time of lactoferrin..Conclusion:(1) A large quantity and activity of the good cells were obtained by mixed enzymedigestion method. High purity osteoblasts were obtained by selective platingtechnique that can remove impurities such as fibroblast cells.(2) the low concentration of lactoferrin (<100ug/ml) was in time dependent topromote the osteoblast proliferation, and high concentration of lactoferrin (>100ug/ml) inhibit the proliferation of osteoblast shows the time dependence.