降钙素基因相关肽对大鼠骨髓单核巨噬细胞破骨分化作用的实验研究  

The effect of calcitonin gene-related peptide on osteoclast differentiation by rat bone marrow macrophages in vitro

作　　者：张文强[1] 段春光[1] 孟国林[1] 王春梅[1] 唐鹏[1] 王铮[1] 曾艳平[1] 刘建[1] 

机构地区：[1]第四军医大学西京医院骨科,西安710032

出　　处：《中国骨质疏松杂志》2015年第4期403-408,共6页Chinese Journal of Osteoporosis

基　　金：国家863项目(2012AA020502-6)

摘　　要：目的通过体外实验研究外源性降钙素基因相关肽(calcitonin gene-related peptide,CGRP)对大鼠骨髓单核巨噬细胞(BMMs)破骨分化能力的影响,为CGRP在抗骨质疏松治疗方面的应用提供理论依据。方法采用差速贴壁的方法分离出SD大鼠髓腔内单核巨噬细胞,原代培养并传代,在培养体系中添加含不同浓度CGRP(实验组:10-7mol/L、10-8mol/L、10-9mol/L;对照组:无CGRP)的破骨诱导液,对前体细胞进行破骨诱导7天后进行相关实验检测,分别采用抗酒石酸酸性磷酸酶染色(TRAP染色)观察破骨细胞的生成能力;用RT-PCR方法检测破骨细胞系特征性基因(RANK、TRAP、NFATc1);Western-blot检测破骨特征性TRAP和RANK蛋白的表达;骨磨片甲苯胺蓝染色检测诱导的破骨细胞的骨吸收功能。结果 TRAP染色显示CGRP各浓度组镜下成熟破骨细胞的个数显著低于对照组,有统计学差异(P<0.05)并随CGRP浓度的增高成熟破骨细胞数减少,CGRP组间亦差异明显(P<0.05);RT-PCR检测破骨特征性RANK、TRAP、NFATc1 mRNA和Western-blot测破骨特征性TRAP、RANK蛋白的表达各CGRP组均低于对照组(P<0.05);骨磨片破骨细胞甲苯胺蓝染色后单倍视野下CGRP组破骨陷窝数量也明显少于对照组(P<0.05),且与药物组浓度与陷窝数量呈负相关性。结论 CGRP能够抑制BMMs向破骨方向的分化,为CGRP抗骨质疏松的治疗提供理论支持。Objective To investigate the effect of calcitonin gene-related peptide （ CGRP） on the osteoclast differentiation by rat bone marrow macrophages （ BMMs ） in vitro, and to find evidence of CGRP in the application of anti-osteoporosis treatment. Methods BMMs were isolated from rats by differential adhesion and attachment method and subcultured.Different dosages of CBRP （ experimental group： 1 ×10 -7 mol/L, 1 ×10 -8 mol/L, 1 ×10 -9 mol/L of CGRP; control group： no CGRP ） were additioned to the culture medium for osteoclast differentiation for 7 days.TRAP staining was performed to estimate the formation of osteoclasts.RT-PCR was used to detect the expression levels of osteoclast specific genes, RANK, TRAP, and NFATc1.The expression of TRAP and RANK protein were detected using Western blotting.Toluidine blue staining of lacunar resorption pits were performed to detect the osteoclast function.Results Compared with that in control group, the number of osteoclasts reduced in a dose-dependent manner in experimental groups （ P〈0.05 ） .RT-PCR results showed that the expression levels of osteoclast gene markers in CGRP groups were significantly lower than that in control group （ P〈0.05 ） .Western blotting results showed that the protein levels of TRAP and RANK were lower in experimental groups than in control group.Toluidine blue staining showed that the lacunar resorption pits in CGRP groups were obviously less than those in control group （ P〈0.05 ） , and a negative correlation between the CGRP concentration and pits quantity was also shown.Conclusion CGRP inhibits osteoclast differentiation by BMMs.This provides a theoretical support for the treatment of osteoporosis.

关 键 词：降钙素基因相关肽 单核巨噬细胞 破骨分化 

分 类 号：R681[医药卫生—骨科学]