破骨细胞在脂多糖介导骨丢失小鼠中的作用及机制研究

Role and mechanism of osteoclasts in lipopolysaccharide-induced bone loss in mice

目的 探讨脂多糖(lipopolysaccharide,LPS)通过刺激T细胞从而诱导破骨细胞分化增殖的作用机制。方法 取雌性C57bl/6小鼠10只,随机分为两组:LPS组(n=5)按2.5 mg/kg隔天腹腔注射LPS,对照组(Con组,n=5)注射等量生理盐水,共计28天。之后处死所有小鼠,收集股骨,应用micro-CT扫描分析小鼠股骨骨密度、骨体积分数、骨小梁数目、骨小梁厚度、骨小梁空隙,眼球取血测炎症因子白介素-17A(interleukin-17A,IL-17A)的表达量,取两组小鼠的骨髓血进行流式细胞术,分析Th17细胞亚群比例。在体外将Th17肿瘤坏死因子α^(+)(tumor necrosis factor α^(+),TNF-α^(+))细胞和CD4^(+)IL-17^(-)T细胞分别与成骨细胞、单核细胞体系以及RAW264.7细胞体系共混培养,观察破骨细胞的增殖分化情况。结果 LPS组小鼠外周血炎症因子IL-17A的表达量显著高于Con组[(1.460±0.395) pg/ml vs.(0.859±0.054) pg/ml],差异有统计学意义(P<0.01)。LPS组骨髓血中Th17 TNF-α^(+)细胞比例显著高于Con组[(2.624±0.783)%vs.(0.35±0.026)%],差异有统计学意义(P<0.01)。在成骨细胞、单核细胞体系中Th17 TNF-α^(+)组的破骨细胞分化的数量显著高于CD4^(+)IL-17^(-)T细胞组(483.97±60.92 vs.158.21±32.56),差异有统计学意义(P<0.05),Th17TNF-α^(+)组破骨细胞F-actin环荧光强度显著强于CD4^(+)IL-17^(-)T细胞组(5.28±0.64 vs.1.79±0.23),差异均有统计学意义(P<0.05)。在RAW264.7细胞体系中Th17 TNF-α^(+)组的破骨细胞分化的数量显著高于CD4^(+)IL-17^(-)T细胞组(322.33±43.35 vs.90.72±15.05),差异有统计学意义(P<0.05),Th17 TNF-α^(+)组破骨细胞F-actin环荧光强度显著强于CD4^(+)IL-17^(-)T细胞组(4.07±0.32 vs.1.00±0.17),差异均有统计学意义(P<0.05)。结论 LPS能刺激骨髓中Th17TNF-α^(+)细胞亚群的比例增高,进而促进破骨细胞的增殖分化。

Objective To investigate the mechanism of lipopoly saccharide(LPS) inducing osteoclast differentiation and proliferation by stimulating T cells.Methods Ten female C57b1/6 mice were randomly divided into two groups.The experimental group(n=5) was intraperitoneally injected with LPS(2.5 mg/kg) every other day for 28 days,and the control group(n=5) was injected with the same amount of normal saline.Bone mineral density,bone volume fraction,trabecular number,trabecular thickness,and trabecular space were analyzed by micro-CT scanning.The expression of inflammatory factor interleukin-17A(IL-17A) was measured by eyeball blood.Flow cytometry was used to analyze the proportion of Th17 cells in bone marrow blood of the two groups.Th17 TNF-α^(+)cells and CD4^(+)IL-17~-T cells were co-cultured with osteoblasts,monocytes and RAW264.7 cells in vitro to observe the proliferation and differentiation of osteoclasts.Results The expression of IL-17A in peripheral blood of LPS group(1.460 ± 0.395) pg/ml was significantly higher than that of Con group(0.859 ± 0.054) pg/ml,and the difference was statistically significant(P<0.01).The proportion of Th17 TNF-α^(+) cells in bone marrow blood of LPS group(2.624 ± 0.783) % was significantly higher than that of Con group(0.35 ā 0.026)(P0.01).The number of osteoclast differentiation in the Th17 TNF-α^(+) group(483.97 ± 60.92) was significantly higher than that in the CD4^(+)IL-17~-T cell group(158.21 ± 32.56)(P<0.05).The fluorescence intensity of F-actin ring in osteoclasts of Th17 TNF-α^(+) group(5.28 ± 0.64) was significantly stronger than that of CD4^(+)IL-17~-T group(1.79 ± 0.23)(P0.05),and the differences were statistically significant.In RAW264.7 cells,the number of osteoclast differentiation in Thl7 TNF-α^(+) group(322.33 ± 43.35) was significantly higher than that in CD4^(+) IL-17~-T group(90.72 ± 15.05)(P<0.05).The fluorescence intensity of F-actin ring in osteoclasts of Th17 TNF-α^(+) group(4.07 ± 0.32) was significantly stronger than that of CD4^(+)IL-17~-T group(1.00 ± 0.17)(P<0.05),and the differences were statistically significant.Conclusions LPS can increase Th17 TNF-α^(+) cells in bone marrow,and then promote the proliferation and differentiation of o steoclasts.