Bovine Lactoferrin Improve Osteoblast Proliferation And Differentiation Via The IGF-Ⅰ/IGFBPs Pathway

Objective:The objective of this study was to evaluate the effect of lactoferrin (LF) onproliferation, differentiation and IGF-Ⅰ,IGFBP2gene expression of primary ratosteoblast in vitro, to explore whether lactoferrin is time and dose-dependent topromote osteoblast proliferation and differentiation, and the molecular mechanism oflactoferrin on anti-osteoporosis and to provide the experiment basis for clinical druguse.Methods:(1) Primary rat osteoblasts were isolated by mixed enzyme which contains trypsinand collagenaseⅠfrom neonatal rat calvarial bone and identified using alkalinephosphatase dyeing and collagen Ⅰ immunohistochemical staining.(2) With theintervention of different concentrations of lactoferrin on osteoblasts, the experimentalgroups was divided into the following:Control group, LF1(0.1ug/ml), LF2(1ug/ml),LF3(10ug/ml), LF4(100ug/ml), LF5(1000ug/ml).The cell proliferation anddifferentiation are respectively assayed by MTT and PNPP,while the gene expressionof IGF-ⅠmRNA and IGFBP2mRNA was measured by Real-Time fluorescencequantitative PCR after1,3,5,7day.(3) IGF-ⅠmRNA expression was knocked downby micro RNA interfering technique.(4)Osteoblasts with the silencing of IGF-Ⅰwere interfered by lactoferrin,and then the cell proliferation and differentiation werealso assayed by using MTT and PNPP methods.(5)Statistical methods: Experimentaldata using mean±standard deviation to show, SPSS16.0statistical software is appliedfor statistical analysis.The groups were compared using one-way analysis of variance(ANOVA),there was statistically significant when P﹤0.05.Results:(1)The cultured cells have the typical features of osteoblast.Alkaline phosphatasedyeing and collagenⅠimmunohistochemical staining are both positive.(2) The resultof MTT method shows that the medication groups’OD value except LF1group are greater than the control group, and rising along with the increasing of theconcentration and processing time of lactoferrin. The medication groups except LF1are greater than the control group(p<0.01) after1day.LF2、LF5have statisticallysignificant differences (P<0.05) with control group, while LF3、LF4is much greaterthan the control(p<0.01) after3days.LF2、 LF3have statistically significantdifferences (P<0.05) with control group while LF4is much greater but LF5is muchlower than the control(p<0.01) after5days.After7days, LF2is greater but LF5ismuch lower than the control (p<0.01),although LF3and LF4are greater than thecontrol but there is no statistically significance.(3) The result of ALP activity showsthat the medication groups is greater than the control group, and rising along with theincreasing of the concentration and processing time of lactoferrin.All of LF groupsexcept LF1have effect on ALP activity of osteoblasts. The ALP activity of LF5reachthe most highest after7days.(4). The result of real-time fluorescence quantitativepolymerase chain reaction (PCR) shows that treatment with10ug/ml,100g/ml,1000ug/ml LF caused a significant increase in the IGF-Ⅰ mRNAlevels but a significance decrease in the IGFBP2as compared with the Con group(P<0.01) and this effect is time and dose-dependent.(5)The results of Real-time PCRand western blot identify that IGF-Ⅰwas knock down.(6)With the silencing of IGF-Ⅰ,the cell proliferation and differentiation of osteoblasts treated with or withoutlactoferrin intervention are both suppressed with control group(P<0.01).There is nosignificance between NC and control group,and so does shRNA group andLF+shRNA group(p>0.05).Conclusion:（1）A large quantity and activity of the good cells were obtained by mixedenzyme digestion method. High purity osteoblasts were obtained by selective platingtechnique that can remove impurities such as fibroblast cells.（2）Lactoferrin is timeand dose-dependent to promote the osteoblast proliferation and differentiation.(3)Lactoferrin can promote IGF-Ⅰ but inhibit IGFBP2gene’s expression inosteoblasts.(4)The proliferation and differentiation of primary rat osteoblasts inresponse to lactoferrin are mediated in part by increased IGF-1gene expression.