The Effects Of Lactoferrin On Proliferation And Expressions Of IGF-1/IGF-1R MRNA In Rat Primary Osteoblasts

Objective: To investigate the effects of lactoferrin on proliferation and expressions ofIGF-1/IGF-1R mRNA in rat primary osteoblasts.Methods: Osteoblasts were separated by multienzyme digestion methods from the cranial bones of6newborn SD rats cultured. Lactoferrin of the concentrations of0、0.1、1、10、100、1000ug/mlwere administrated into the culture medium for1、3、5、7days. The proliferation of osteoblasts wasdetected by3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide test(MTT). ThemRNA expression of Insulin-like growth factor1(IGF-1) and Insulin-like growth factor1receptor(IGF-1R) were examined by realtime reverse transcription-polymerase chain reaction(Real time-PCR) respectively.Results：（1）The cultured cells have the typical features of osteoblast. The cells aretriangle, polygonal, long spindle shape, etc, and mostly are mononuclear cells, havelarge pseudopodia. Alkaline phosphatase dyeing collagenⅠimmunohistochemicalstaining are both positive.(2) Compared with the blank control group, the proliferation rates of the osteoblastsexposed to the lactoferrin of the concentrations of0.1ug/ml were not statisticallysignificant (P>0.05). The concentrations of1、10、100、1000ug/ml for1、3、5、7dayswere all higher than those of the blank control group(P<0.05), and the concentrations of1、10、100、1000ug/ml were statistically significant (P<0.01). As time goes on, thenumber of osteoblasts were increased exposed to different concentrations of lactoferrin,the number of osteoblasts got the peak at the100ug/ml in the7th days.(3) The IGF-1mRNA expression of the osteoblasts exposed to10、100、1000ug/mllactoferrin were higher compared with the control group in each time (P<0.05), and theconcentrations of10、100ug/ml were statistically significant (P<0.01). As time goes on, The IGF-1mRNA expression of osteoblasts were increased exposed to differentconcentrations of lactoferrin. Various concentration were significantly higher comparedwith the blank control group in5、7day (P<0.01).（4）The IGF-1R mRNAexpression of the osteoblasts exposed to10、100、1000ug/mllactoferrin were higher compared with the control group in each time (P<0.05), and theconcentrations of10、1000ug/ml were statistically significant (P<0.01). As time goeson, The IGF-1R mRNA expression of osteoblasts were increased exposed to differentconcentrations of lactoferrin in3、5、7day compared with the control group（P<0.05）.Various concentration were significantly higher compared with the blank control groupin5、7day (P<0.01).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 can increase the proliferation of the osteoblasts Lactoferrin also canincrease the expression of IGF-1/IGF-1R mRNA expression in rat primary osteoblasts.It may be one of the mechanism of lactoferrin prevention and cure of Osteoporosis.