The Effect Of Calcitonin Gene-Related Peptide On Osteoclasts Differentiation And Function Of Rat Bone Marrow Macrophages In Vitro

In clinical treatment process, we often found that patients of traumatic brain injured,with limb fractures tend to have an overgrowth even heterotopic ossification at the fracture site, and the healing time was less than those who only sufferd limb fractures. On the other hand, a fractured patient who hurts his peripheral nerve often got higher possibility of bone ununion or delayed.The earlier researchs showed that the levels of some neuropeptides in the patients blood who suffered central nerve damage could got higher, that may be the reason why bone callus of fracture patients got excess growth. These neuropeptides includ calcitonin gene-related peptide(CGRP) and substance P(SP), etc. Obviously, It is well known that CGRP which could widely distribute in the body and accelerate bone formation has been shown to play an important role in regulating bone homeostasis. However, it is not clear that what is the CGRP’s role to BMMs in bone resorption. To further verify the reasons of acceleration mechanisms of bone formation, we observe and investigate the effects of exogenous CGRP on BMMs from the perspective of bone resorption, which may provide new insights into fractures in terms of treatment.Part1. Effect of calcitonin gene-related peptide on osteoclasts differentiation of rat bone marrow macrophages cellsObjective:: To explore the effect of calcitonin gene-relate peptide on the osteoclasts differentiation of rat bone marrow macrophages cells. Methods:(1)BMMs were isolated from rats by differential adhesion and attachment methods.The s RANKL +M-CSF culture media were adopted to culture osteoclasts from BMMs.(2)Different dosages of calcitonin gene-related peptide(A:the control group :excluding CGRP; B: the high concentration of CGRP group:1×10-7 mol/L; C: the medium concentration of CGRP group :1× 10-8mol/L; D: the low concentration of CGRP group:1× 10-9 mol/L)were added to the culture medium for the observation of osteoclast differentiation.(3)TRAP staining was observed to estimate the formation of osteoclasts and count the number of osteoclasts cultured by different doses of CGRP at Day 7.(4) RT-PCR was used to detect the expression level of RANK、TRAP、NFATc1 m RNA of osteoclasts cultured by different doses of CGRP at Day 7.(5)the protein level of TRAP and RANK of osteoclasts cultured by different doses of CGRP were detected by Western-blot at Day 7. Results:(1)Compared with control group,the number of osteoclasts of CGRP group reduced in a dose-dependent manner in TRAP staining(p<0.05).(2)The expression level of RANK、TRAP、NFATc1 m RNA of CGRP groups were significantly lower than control group(p < 0.05). Further more, Western-blot proved this results: the expressions of TRAP and RANK were less than control group too(p < 0.05). Conclusions:The osteoclasts differentiation of the BMMs are restrained by CGRP, which may play an important role in bone repair and reconstruction.Part2. Effect of calcitonin gene-related peptide on osteoclasts function of rat bone marrow macrophages cellsObjective: To explore the effect of calcitonin gene-relate peptide on the osteoclasts function of rat bone marrow macrophages cells. Methods:(1) BMMs were isolated from rats by differential adhesion and attachment methods. The s RANKL +M-CSF culture media were adopted to culture osteoclasts from BMMs.(2) Different dosages of calcitonin gene-related peptide(A:the control group :excluding CGRP; B: the high concentration of CGRP group:1×10-7 mol/L; C: the medium concentrationof CGRP group :1× 10-8mol/L; D: the low concentration of CGRP group:1× 10-9 mol/L)were added to the culture medium for the observation of osteoclast differentiation.(3)TRAP staining was observed to estimate and identify the formation of osteoclasts cultured by excluding CGRP at Day 5and 7.(4) RT-PCR was used to detect the expression level MMP-9,Cathepsin K m RNA of osteoclasts cultured by different doses of CGRP at Day 7.(5) Toluidine blue staining of lacunar resorption pits were performed to detect the function of osteoclasts cultured by different doses of CGRP at Day 7. Results:(1)Compared with control group,the proliferation of the BMMs are restrained by CGRP; with the increase of concentration of CGRP, inhibitory effect was more apparent.(2)The expression level of MMP-9,Cathepsin K m RNA of CGRP groups were significantly lower than control group(p < 0.05).(3) Toluidine blue staining showed that the lacunar resorption pits of CGRP groups were obviously less than control group(p < 0.05), and a negative correlation between the CGRP concentration and pits quantity was also showed in our experiment. Conclusions:The osteoclasts function of the BMMs were restrained by CGRP,which may provides an important theoretical basis for clinical treatment.