中国修复重建外科杂志

中国修复重建外科杂志

脂多糖对破骨细胞生成及骨吸收功能作用及其机制研究

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目的 探讨脂多糖(lipopolysaccharide,LPS)对破骨细胞生成及其吸收功能的作用及机制。 方法 取雄性 C57BL/6 小鼠股骨及胫骨骨髓,分离培养骨髓源巨噬细胞(bone marrow-derived macrophages,BMMs),并行流式细胞仪鉴定。取 BMMs 采用不同浓度 LPS(0、100、200、500、1 000、2 000 ng/mL)培养后,以细胞计数试剂盒 8(cell counting kit 8,CCK-8)检测不同浓度 LPS 对细胞活性影响。为探讨 LPS 对破骨细胞生成的影响,取 BMMs 分为巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)组、M-CSF+核因子 κB 受体活化因子配体(receptor activator of nuclear factor κB ligand,RANKL)组、M-CSF+RANKL+50 ng/mL LPS 组、M-CSF+RANKL+100 ng/mL LPS 组,对应培养后行抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphates,TRAP)染色观察,计算破骨细胞面积百分比。为探讨 LPS 对 Connexin43 蛋白及基因表达影响,将 BMMs 分别分为对照组(M-CSF+RANKL)、LPS 组(M-CSF+RANKL+100 ng/mL LPS)以及对照组(M-CSF+RANKL)、50 ng/mL LPS 组(M-CSF+RANKL+50 ng/mL LPS)、100 ng/mL LPS 组(M-CSF+RANKL+100 ng/mL LPS)培养后,行 Western blot 以及实时荧光定量 PCR 检测。为探讨 LPS 对破骨细胞骨吸收能力的影响,将 BMMs 分为 M-CSF 组、M-CSF+RANKL 组、M-CSF+RANKL+50 ng/mL LPS 组、M-CSF+RANKL+100 ng/mL LPS 组对应培养后,采用骨吸收实验检测骨吸收面积百分比。 结果 流式细胞仪鉴定培养细胞为 BMMs。CCK-8 法检测显示与其他浓度相比,100 ng/mL LPS 明显促进 BMMs 活性(P<0.05)。TRAP 染色示,M-CSF 组未见破骨细胞生成;与 M-CSF+RANKL 组相比,M-CSF+RANKL+50 ng/mL LPS 组、M-CSF+RANKL+100 ng/mL LPS 组破骨细胞体积更大、细胞核更多,其中后者最显著,3 组破骨细胞面积百分比差异均有统计学意义(P<0.05)。Western blot 检测,LPS 组 Connexin43 蛋白相对表达量较对照组明显提高(P<0.05);实时荧光定量 PCR 检测示,对照组、50 ng/mL LPS 组以及 100 ng/mL LPS 组 Connexin43 基因相对表达量逐渐增加,比较差异有统计学意义(P<0.05)。骨吸收实验示,M-CSF 组未形成破骨细胞骨吸收;M-CSF+RANKL 组、M-CSF+RANKL+50 ng/mL LPS 组、M-CSF+RANKL+100 ng/mL LPS 组骨吸收面积百分比逐渐增加,比较差异均有统计学意义(P<0.05)。 结论 100 ng/mL LPS 能够促进 Connexin43 的表达,从而使破骨细胞生成增多,骨吸收功能加强。

Objective To study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function. Methods Bone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate-resistant acid phosphates (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area. Results The flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant difference when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS (P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant (P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group (P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant (P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant (P<0.05). Conclusion LPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.

关键词: 破骨细胞; 骨髓源巨噬细胞; 脂多糖; 缝隙连接蛋白 43; 小鼠

Key words: Osteoclasts; bone marrow-derived macrophages; lipopolysaccharide; Connexin43; mouse

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