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目的建立破骨细胞(osteoclasts,OCs)体外分化成熟的模型,研究CD147对OCs分化成熟的影响。方法从外周血分离单个核细胞贴壁培养,使用破骨细胞分化因子(receptor activator of nuclear factor-κβligand,RANKL)和巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)诱导OCs生成。实验分为对照组、诱导组(RANKL+M-CSF)及阻断组(RANKL+M-CSF+CD147Ab),应用免疫荧光染色和流式细胞术检测CD147在破骨前体细胞(osteo-clast precursor cells,OPCs)的表达;抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色观察OCs的分化成熟情况及Real-time PCR技术检测CD147及TRAP mRNA在OPCs中的表达变化。结果①CD147分子在OPCs细胞膜上表达。②流式细胞术测得细胞膜表面平均荧光强度对照组(159.55±1.65)、诱导24 h组(171.18±3.46)、诱导48 h组(178.47±4.59)逐渐增强(P<0.05),而阻断24 h组(117.69±9.01)显著减弱(P<0.01)。③阻断组TRAP染色阳性细胞(细胞核≥3)数显著少于诱导组(P<0.01),且OCs体积小。④在24、48 h诱导组CD147 mRNA相对表达量[(1.393±0.248)、(1.772+0.326)]及TRAP mRNA的相对表达量[(1.810±0.398)、(2.608±0.906)]在OPCs诱导分化过程逐渐增高,均高于相应时相点阻断组(P<0.05);且TRAP mRNA相对表达量与CD147 mRNA相对表达量呈正相关(r=0.833,P<0.01)。结论 CD147分子在OPCs细胞膜上表达且参与OCs分化成熟;抑制CD147的表达,OCs出现成熟障碍。
Objective To establish a model of differentiation and maturation of osteoclasts (OCs) in vitro and investigate the effect of CD147 on differentiation and maturation of OCs. Methods Peripheral blood mononuclear cells were isolated and cultured. The OCs were induced by using receptor activator of nuclear factor-κβ ligand (RANKL) and macrophage colony stimulating factor (M-CSF). The experiment was divided into control group, induction group (RANKL + M-CSF) and blocking group (RANKL + M-CSF + CD147Ab). Immunofluorescence staining and flow cytometry were used to detect the expression of CD147 in osteo-clast The expression of CD147 and TRAP mRNA in OPCs were detected by real-time PCR. The expression of CD147 and TRAP mRNA in OPCs was detected by tartrate-resistant acid phosphatase (TRAP) staining. Results ①CD147 was expressed on the membrane of OPCs. Flow cytometry showed that the average fluorescence intensity of the cell membrane surface was increased (159.55 ± 1.65), induced for 24 h (171.18 ± 3.46) and induced for 48 h (178.47 ± 4.59) (P <0.05) 24 h group (117.69 ± 9.01) significantly weakened (P <0.01). ③ The numbers of TRAP-positive cells (nucleus≥3) in blocking group were significantly less than those in induction group (P <0.01), and the volume of OCs was small. ④ The relative expression of CD147 mRNA in induced group [(1.393 ± 0.248), (1.772 ± 0.326)] and TRAP mRNA in induced group [(1.810 ± 0.398), (2.608 ± 0.906) (P <0.05). The relative expression of TRAP mRNA was positively correlated with the relative expression of CD147 mRNA (r = 0.833, P <0.01). Conclusion The CD147 molecule is expressed on the membrane of OPCs and participates in the differentiation and maturation of OCs. The inhibition of CD147 expression and the maturation of OCs appear.