Lactoferrin Effected The Expression Of IGFBP-3,IGFBP-4 And IGFBP-5 In Primary Rat Osteoblasts

Objective To research weather lactoferrin affects the expression of IGFBP-3,IGFBP-4 and IGFBP-5 of primary rat osteoblasts.Then to provide referrence for the molecular mechanism that how the bone metabolism was effectect by lactoferrinMethods Osteoblasts was harvested from the skull of SD rattus borned in 24 hours by enzyme digestion.And it was identified by alkaline phosphatase and type I collagen immunohistochemical staining.The expression of m RNA of IGFBP-3,IGFBP-4 and IGFBP-5 of primary rat osteoblasts was analyzed by Real-time fluorescent quantitative PCR,while the expression of protein was analyzed by western blotting.Results Compared to the control group,the expression of IGFBP-3 m RNA of primary rat osteoblasts was inhibited(P <0.01)by different concentrations of lactoferrin intervented on 1 day and 3 days.The m RNA of IGFBP-3 in primary rat osteoblasts was inhibited by low concentration groups((0.1ug/ml?1ug/ml?10ug/ml?100ug/ml)on the 5d and 7d,while promoted by 1000ug/ml.The expression of IGFBP-3 protein in primary rat osteoblasts was downregulated by 1000ug/ml on different time periods and was upregulated on 1d and 5d by low concentration groups.Though,The expression of IGFBP-3 protein seems to be downregulated,but it was no more than twice.Compared to the control group,the expression of IGFBP-4 m RNA sustainingly inhibited on 1,3,5,7 days,while at the protein level,not detected.The expression of IGFBP-4 m RNA in primary rat osteoblasts was maily inhibited by different concentrations of lactoferrin.But it was promoted on 3d and the7 d,but no more than twice.The expression of IGFBP-5 protein in primary rat osteoblasts was mainly promoted on the 1d and 5d.While it seemed to be downregulated on 7d,it was lower than twice.There was no significant change on 3d and 7d.Conclusion In this paper,first of all,larger number and higher purity of primary rat osteoblasts was obtained by using hybrid enzyme digestion.Second,the expression of insulin growth factor binding protein-3,insulin growth factor binding protein-4,insulin growth factor binding protein-5 in primary osteoblasts were effected by lactoferrin.Third,the expression of IGFBP-3,IGFBP-5 in primary osteoblasts may be effected by lactoferrin in two different levels and it was unstable in m RNA level and the main effect of lactoerrin in protein level may be positive.The IGFBP-4 was inhibited in a concentration dependent manner which the higher concentration the lower expression would be and IGFBP-4 protein may degradation by enzymatic hydrolysis of PAPP-A.