小鼠巨噬细胞系RAW 264.7向破骨细胞分化的诱导条件

Induction conditions of osteoclasts differentiation from mouse macrophage RAW 264.7 cell line

目的探讨RAW 264.7细胞与向破骨细胞分化的诱导条件,以期提高破骨细胞的数量与活性。方法采用不同浓度核因子κB受体活化因子配体(receptor activator of nuclear factorκB ligand,RANKL)诱导RAW 264.7细胞4 d后形成破骨细胞,并通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色和鬼笔环肽染色鉴定破骨细胞,骨板吸收实验检测破骨细胞的骨吸收活性,反转录酶-聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)检测破骨细胞功能基因TRAP、破骨细胞相关受体(osteoclast-associated receptor,OSCAR)、c-Fos、核转录因子激活的T细胞1(nuclear factor of activated T-cells 1,NFATcl)的表达。结果 RAW 264.7细胞经RANKL诱导4 d后可出现大量TRAP呈阳性和纤维肌动蛋白环形成的多核破骨细胞,骨板上形成较多的骨吸收瘢痕,RT-PCR检测到破骨细胞特殊功能基因信使核糖核酸(message ribonucleic acid,mRNA)高表达。结论 RAW 264.7诱导4 d可产生大量噬骨能力较强的破骨细胞。

Objective The study aimed to explore the induction conditions of RAW 264.7 cells differentiation into osteoclasts,with the view to improving the amount and activity of osteoclasts. Methods The RAW 264.7 cells were induced with different concentrations of receptor activator of nuclear factor κB ligand (RANKL) for 4 days to differentiate into osteoclasts, tartrate-resistant acid phosphatase (TRAP) staining and phalloidin staining were used to identify the osteoclasts, the Osteo Assay Surface Plate was used to measure the bone resorptive activities of osteoclasts, reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expressions of osteoclast phenotypic and functional genes TRAP, osteoclast-associated receptor (OSCAR), c-Fos and nuclear factor of activated T-cells 1 (NFATc1). Results RAW 264.7 cells induced by RANKL 4 days, could differentiate into multinucleated cells and a large number of fibrous actin rings formed by TRAP positive 1fibers and fibrous actin ring; Bone resorption pits were found on the surface of bone plate by microscope; RT-PCR detected that high expression of message ribonucleic acid (mRNA) made by the gene messenger of osteoclasts for special function. Conclusions The RAW 264.7 cells induced for 4 days can differentiate into a large number of osteoclasts with strong bone resorption activity.