| IGF-1 was concerned as the key factor in regulating homeostasis of the bone metabolism and the interaction between osteoblast and osteoclast, beyond RANKL/OPG pathway. There are rhIGF-1R being expressed on the cell membrane of either osteoblast and osteoclast, and both of them could be activated by rhIGF-1. The aim of this article is try to discuss the interact regulating action between osteoblast and osteoclast under the treatment of rhIGF-1.Method1.Osteoclast precursor cell line RAW264.7 was induced differentiation into osteoclasts by RANKL, certificated by TRAP staining.2. rhIGF-150ng/ml were added to osteoblasts and RAW264.7-derived osteoclast respectively, intervention time were:6min,20min,60min. Western-blotting was applied to confirm the activation of rhIGF-1 receptor.3.Rabbit anti-rhIGF-I IgG were added to the medium with the final concentrations of 0,2,4,6 ug/ml, Western-blotting to select the optimized neutralizing concentration of rhIGF-I.4.Re-inoculated osteoblasts and osteoclasts, added 0,10,50,100 ng/ml of rhIGF-1 respectively to interfere osteoblasts and osteoclasts, osteoblast-conditioned medium(OBCM) and osteoclast-conditioned medium (OCCM)with rhIGF-1 were collected after 12 hours, then the experiment was divided into three groups:A: rhIGF-1 directly treated groups; B:Conditioned medium cross-cultured groups: Conditioned medium were collected and filtered, then exchanged the medium and continue to culture OB and OC for 12h; C:Conditioned medium with rhIGF-1 neutralization cross-cultured groups:conditioned medium were collected and added 4ug/ml anti-rhIGF-I IgG, exchanged the medium and continue to culture OB and OC for 12h.(1) RANKL,OPG and BGP in the medium were examined by ELISA.(2) Bgp,Opg,Rankl mRNA in OB and Ck,Mmp-9,Rank mRNA in OC were quantitatively determined by Real-time polymerase chain reaction.Result 1. Under the induction of RANKL, with 50ng/ml for 8 days,RAW264.7 cells were induced into multiple-nucleus osteoclasts.2.Western-blotting results confirmed that rhIGF-I could result in rhIGF-1R phosphorylation in both OB and OC.And the amount of phosphorylated rhIGF-1R were increased as the interfere time extended.3. Western-blotting results confirmed that the amount of phosphorylated rhIGF-1R were does dependtly decreased in the presence of anti-rhlGF-â… .And when anti-rhIGF-â… reached 4,6ug/ml, activated rhIGF-1 receptor ocould not be detected4.(1) After OB were treated with different dose of rhIGF-1, to examined the content of RANKL and BGP in OBCM by ELISA,and the results showed that there were no significant difference among different groups.However, when OB cultured with OCCM and OCCM after antibody neutralizing, we found that three cell factors expression were significantly higer than other groups in the presence of rhIGF-1 at 10,50ng/ml, it was highest rhIGF-1 at 10ng/mL; In low dose group.the three cytokines in C group were significantly higher than that in B group.(2) Real-time PCR results showed that in A group, the mRNA expression levels of Bgp,Opg,Rankl in OB and Ck,Mmp-9,Rank in OC were no significant difference among different groups; However, there were significantly higher than other dose of groups when rhIGF-1 reached 10ng/ml. Statistical analysis showed that the differences is significant (p<0.05).ConclusionIn the conclusion, results above showed that when different dose of rhIGF-1 were added to OB and OC, the effect were not significant; However, when cultured OB with OCCM and rhIGF-1 neutralized OCCM or cultured OC with OBCM and rhIGF-1 neutralized OBCM, the changes were very significant. It was the most obvious in the low dose group. Therefore, our study suggested that rhIGF-1 may primarily caused the change of OC, then alterted OC activities and its micro-environmental changement subsequently affected the growth and functions of OB. Which might be happened same to the OC. |
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