破骨细胞参与时IGF-1对成骨细胞的促同化作用  

The anabolic effect of IGF-1 on the osteoblast in the presence of the osteoclast

作　　者：赵荣兰[1] 彭效祥 李俏俏[1] 窦烨[1] 孙蓓[3] 

机构地区：[1]山东万杰医学院,生化教研室,淄博255213 [2]山东万杰医学院,微生物与免疫教研室 [3]天津医科大学代谢病医院内分泌研究所卫生部激素与发育重点试验室

出　　处：《中国骨质疏松杂志》2010年第12期907-912,共6页Chinese Journal of Osteoporosis

摘　　要：目的探讨IGF-1对成骨细胞(osteoblast,OB)的促同化作用是否需要破骨细胞(Osteoclast,OC)的协同。方法体外培养MC3T3小鼠成骨细胞及RAW264.7小鼠单核巨噬细胞,RAW264.7细胞经核因子κB受体活化因子配基(Receptor activator of nuclear factor kappa-B ligand,RANKL)诱导分化为破骨细胞。以50 ng/ml重组人胰岛素样生长因子-1(rhIGF-1)分别干预成骨细胞及分化成熟的破骨细胞,Wstern blotting验证IGF-1受体的活化。以0、10、50、100 ng/ml的rhIGF-1分别干预成骨细胞、破骨细胞及共培养的成、破骨细胞。12 h后终止培养,收集破骨细胞经IGF-1干预后的条件培养基(OC conditioned medium after IGF-1 treatment,OCCM)对另一组新接种的成骨细胞干预12 h。ELISA检测3组成骨细胞培基中骨钙素(bone Gla protein,BGP)、RANKL的含量,Real-time PCR检测成骨细胞中Bgp基因的表达。结果 RANKL诱导培养8 d后RAW264.7细胞形成TRAP阳性,成熟的多核破骨细胞;Wstern blotting检测表明rhIGF-1可有效得使成、破骨细胞中的IGF-1受体发生磷酸化;ELISA与Real-time PCR测定结果显示,IGF-1直接干预OB时,Bgp基因表达水平和培养基中BGP及RANKL蛋白的含量均与无干预的对照组无明显区别;而OCCM干预及共培养的OB组,当IGF-1初始干预浓度为10 ng/ml时BGP、RANKL含量及Bgp基因的表达水平显著提高,共培养组与OCCM培养组间无明显区别。结论 IGF-1对OB的促同化作用依赖于OC的参与,两种细胞间的联系可能是通过可溶性的细胞因子来完成。Objective To study whether the osteoclast is necessary for the anabolic effect of IGF-1 on the osteoblast in vitro. Methods Mouse MC3T3 osteoblast cells and RAW264.7 monocyte/macrophage cells were cultured in vitro. Receptor activator of nuclear factor kappa-B ligaod （RANKL） was added to the RAW264.7 cell medium to induce the osteoclast differentiaion. Recombinant human insulin-like growth factor-1 （rhIGF-1） 50 ng/ml were subjected to the treatment of osteoblasts and RAW264. 7-derived osteoclasts, respectively. IGF-1 receptor activation was verified using Western blotting. The cultures of osteoblasts, osteoclasts, or the co-cultures of osteoblasts and osteoclasts were treated with O, 10, 50, 100 ng/ml of rhIGF-1 , respectively. Osteoclast-conditioned medium with rhIGF-1 were collected after a 12-hour treatment. The conditioned medium was applied to the newly cultured osteoblasts for 12 hours. The concentrations of bone Gla protein （BGP） and RANKL in collected media were determined using ELISA. Bgp mRNA in the osteoblast was detected using real-time PCR. Results RAW264. 7 cells could be successfully induced to mature multiple-nucleus and TRAP-positive osteoclasts by RANKL. Western blotting results confirmed that rhlGF-1 could effectively activate IGF-1 receptor phospharylation in both osteoblasts and osteoclasts. Results of ELISA and real-time PCR showed that there were no significant difference in either Bgp gene expression or BGP and RANKL concentrations in the medium between the IGF-l-additioned osteoblasts and the control cells. However, BGP and RANKL concentrations and the Bgp gene expression level were significantly elevated in both osteoblast ceils cultured with osteoclast-conditioned medium and co- cultured cells. There was no significant difference between the osteoclast-conditioned culture group and the co-culture group. Conclusion Osteoclast is necessary for anabolic effect of IGF-1 on cultured osteoblast. The connection between the two cells may be completed through soluble cvtokines.

关 键 词：RANKL 破骨细胞 成骨细胞 IGF-1 

分 类 号：R681[医药卫生—骨科学]