内源性甲状旁腺激素对体外诱导培养破骨细胞的影响
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国家自然科学基金(30973030)


Effect of endogenous parathyroid hormone on osteoclast cultured in vitro
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    摘要:

    目的:研究内源性甲状旁腺激素(parathyroid hormone,PTH)对于体外诱导培养破骨细胞的影响-方法:取8周龄PTH+/+和PTH-/- 小鼠各16只,分离培养小鼠股骨全骨髓细胞,在含有10 ng/ml 巨噬细胞集落刺激因子(M-CSF)的培养皿中培养24 h,收集未贴壁细胞,即得破骨细胞前体,利用M-CSF和核因子κB受体活化因子配体(RANKL)刺激形成破骨细胞-根据加入不同RANKL浓度分为低浓度(50 ng/ml)和高浓度(100 ng/ml)组-于诱导第6天-第9天进行抗酒石酸酸性磷酸酶(TRAP)染色,观察破骨细胞形态并计数40或100倍视野内TRAP阳性细胞数量-结果:M-CSF和RANKL能够在体外诱导小鼠全骨髓细胞形成TRAP阳性的破骨细胞-诱导第9天进行TRAP染色,同一个视野中形成的TRAP阳性破骨细胞数量较诱导第6天减少,但是破骨细胞体积和细胞核数量增多-相同浓度RANKL刺激下,PTH+/+与PTH-/-组破骨细胞数量未见明显统计学差异-不同浓度RANKL刺激下,高浓度组破骨细胞数量明显多于低浓度组细胞数量-结论:内源性PTH对于体外M-CSF和RANKL诱导骨髓培养的破骨细胞数量没有影响,与低浓度组相比,高浓度RANKL能够进一步促进破骨细胞数量增加-

    Abstract:

    Objective:To study the effect of endogenous parathyroid hormone (PTH) on osteoclasts (OCs) cultured in vitro. Methods:Sixteen PTH+/+ and sixteen PTH-/- mice with 8-week-old were used in this study. Bone marrow stem cells obtained from mouse femurs were isolated and cultured with macrophage colony-stimulating factor (M-CSF,30 ng/ml) and different concentration of receptor activator of NF-κB ligand (RANKL,50 ng/ml as low concentration group or 100 ng/ml as high concentration group) in culture dishes containing 10 ng/ml M-CSF for 24 h. The tartrate-resistant acid phosphatase (TRAP) staining was performed to observe and count cell morphology and TRAP-positive osteoclasts numbers at 40 or 100 magnified visual field after 6-day and 9-day culturing. Results:M-CSF and RANKL induced bone marrow stem cells to form TRAP-positive OCs. TRAP-positive OCs numbers in the same visual field decreased after 9-day culturing compared with that after 6-day culturing,but OCs volume after 9-day culturing became more larger and had more nuclei than after 6-day culturing. In the same concentration of RANKL,no changes were observed in the numbers of TRAP-positive OCs between the PTH+/+ groups and the PTH-/- groups. In contrast,either in the PTH+/+ or the PTH-/- groups,high concentration of RANKL (100 ng/ml) induce more TRAP-positive OCs. Conclusion:Endogenous PTH had no effect on the numbers of osteoclasts cultured in vitro in the presence of M-CSF and RANKL. High concentration of RANKL(100 ng/ml) increased the TRAP-positive osteoclast numbers compared with low concentration (50 ng/ml).

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孙鹏,唐鹏宇,凡进,张建伟,张宁.内源性甲状旁腺激素对体外诱导培养破骨细胞的影响[J].南京医科大学学报(自然科学版),2014,(4):467-471

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  • 收稿日期:2013-12-08
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  • 在线发布日期: 2014-04-18
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