小鼠破骨细胞诱导及成纤维生长因子受体表达检测

The Induction of Murine Osteoclasts and the Detection of Fibroblast Growth Factor Receptors

目的:检测成纤维生长因子受体(fibroblast growth factor receptors,FGFRs)在小鼠破骨细胞中的表达情况,为探讨FGFRs对破骨细胞的直接调控作用奠定基础。方法:采用巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)和破骨细胞分化因子(receptor activator of nuclear factor-Bligand,RANKL)诱导小鼠骨髓单核细胞分化为破骨细胞。提取细胞总RNA后经逆转录获得小鼠破骨细胞cDNA,根据FGFRs基因编码区序列设计的引物进行PCR扩增并对PCR扩增产物进行测序。为进一步验证转录水平的结果,提取细胞总蛋白电泳后进行免疫印迹实验。结果:诱导5d后可见TRAP(+)多核细胞出现,小鼠破骨细胞在转录水平和翻译水平均只可检测到FGFR1和FGFR3基因的表达产物。结论:M-CSF和RANKL可成功诱导出小鼠破骨细胞,FGFR1和FGFR3基因在小鼠破骨细胞中均有表达。

Objective： To determine the expression of FGFRs gene in murine osteoclasts for provide the foundation of investigating the relation between the FGFRs expression and biological characters of osteoclasts. Methods： Osteoclasts were induced by bone marrow mononuclear phagocytes of mouse using medium with macrophage colony stimulating factor （M-CSF） and receptor activator of nuclear factor- B ligand （RANKL）.Total RNA was extracted from osteoclasts and cDNA was obtained by inverse transcription. The objective gene was amplified by PCR with primers targeted to nucleotide sequences corresponding to coding regions of FGFRs and the amplified product was sequenced. In order to check the expression of FGFR1 and FGFR3 on translation level, FGFR1 and FGFR3 was detected by western blot. Results： The TRAP positive multinuclear cells were induced from murine bone marrow mononuclear phagocytes after 5 days of inoculating. The results on transcription and translation level show that FGFR1 and FGFR3 were expressed in murine osteoclasts. Conclusion： The osteoclasts were successfully induced from bone marrow mononuclear phagocytes in the presence of M-CSF and RAN- KL. Both FGFR1 and FGFR3 are expressed in murine osteoclasts.